WO2024036377A1 - Hairpin oligonucleotides - Google Patents
Hairpin oligonucleotides Download PDFInfo
- Publication number
- WO2024036377A1 WO2024036377A1 PCT/AU2023/050781 AU2023050781W WO2024036377A1 WO 2024036377 A1 WO2024036377 A1 WO 2024036377A1 AU 2023050781 W AU2023050781 W AU 2023050781W WO 2024036377 A1 WO2024036377 A1 WO 2024036377A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cancer
- hairpin oligonucleotide
- oligonucleotide
- hairpin
- target protein
- Prior art date
Links
- 108091034117 Oligonucleotide Proteins 0.000 title claims abstract description 273
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 192
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 152
- 238000000034 method Methods 0.000 claims abstract description 72
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 67
- 102000040945 Transcription factor Human genes 0.000 claims abstract description 55
- 108091023040 Transcription factor Proteins 0.000 claims abstract description 55
- 201000010099 disease Diseases 0.000 claims abstract description 35
- 208000035475 disorder Diseases 0.000 claims abstract description 32
- 230000027455 binding Effects 0.000 claims description 143
- 125000003729 nucleotide group Chemical group 0.000 claims description 104
- 239000002773 nucleotide Substances 0.000 claims description 97
- -1 CLOCK Proteins 0.000 claims description 78
- 206010028980 Neoplasm Diseases 0.000 claims description 64
- 150000007523 nucleic acids Chemical group 0.000 claims description 62
- 102100038895 Myc proto-oncogene protein Human genes 0.000 claims description 47
- 108091035710 E-box Proteins 0.000 claims description 42
- 201000011510 cancer Diseases 0.000 claims description 33
- 238000012986 modification Methods 0.000 claims description 31
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 30
- 230000004048 modification Effects 0.000 claims description 30
- 230000002401 inhibitory effect Effects 0.000 claims description 28
- 206010006187 Breast cancer Diseases 0.000 claims description 21
- 208000026310 Breast neoplasm Diseases 0.000 claims description 21
- 239000008194 pharmaceutical composition Substances 0.000 claims description 19
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 18
- 239000003814 drug Substances 0.000 claims description 17
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 206010009944 Colon cancer Diseases 0.000 claims description 7
- 239000003085 diluting agent Substances 0.000 claims description 7
- 102100021615 Class A basic helix-loop-helix protein 15 Human genes 0.000 claims description 6
- 102100021307 Cyclic AMP-responsive element-binding protein 3-like protein 4 Human genes 0.000 claims description 6
- 102100027641 DNA-binding protein inhibitor ID-1 Human genes 0.000 claims description 6
- 101000895309 Homo sapiens Cyclic AMP-responsive element-binding protein 3-like protein 4 Proteins 0.000 claims description 6
- 108010050345 Microphthalmia-Associated Transcription Factor Proteins 0.000 claims description 6
- 102100030157 Microphthalmia-associated transcription factor Human genes 0.000 claims description 6
- 102100030589 Neurogenic differentiation factor 6 Human genes 0.000 claims description 6
- 102100040365 T-cell acute lymphocytic leukemia protein 1 Human genes 0.000 claims description 6
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 6
- 102100023033 Cyclic AMP-dependent transcription factor ATF-2 Human genes 0.000 claims description 5
- 101001081590 Homo sapiens DNA-binding protein inhibitor ID-1 Proteins 0.000 claims description 5
- 101001023770 Homo sapiens Transcription factor NF-E2 45 kDa subunit Proteins 0.000 claims description 5
- 206010025323 Lymphomas Diseases 0.000 claims description 5
- 102100023489 Transcription factor 4 Human genes 0.000 claims description 5
- 102100035412 Transcription factor NF-E2 45 kDa subunit Human genes 0.000 claims description 5
- 102100030907 Aryl hydrocarbon receptor nuclear translocator Human genes 0.000 claims description 4
- 102100037211 Aryl hydrocarbon receptor nuclear translocator-like protein 1 Human genes 0.000 claims description 4
- 206010005949 Bone cancer Diseases 0.000 claims description 4
- 208000018084 Bone neoplasm Diseases 0.000 claims description 4
- 102100027309 Cyclic AMP-responsive element-binding protein 5 Human genes 0.000 claims description 4
- 102100027642 DNA-binding protein inhibitor ID-2 Human genes 0.000 claims description 4
- 102100036448 Endothelial PAS domain-containing protein 1 Human genes 0.000 claims description 4
- 102000003817 Fos-related antigen 1 Human genes 0.000 claims description 4
- 108090000123 Fos-related antigen 1 Proteins 0.000 claims description 4
- 102100023855 Heart- and neural crest derivatives-expressed protein 1 Human genes 0.000 claims description 4
- 102100021888 Helix-loop-helix protein 1 Human genes 0.000 claims description 4
- 102100021889 Helix-loop-helix protein 2 Human genes 0.000 claims description 4
- 101000793115 Homo sapiens Aryl hydrocarbon receptor nuclear translocator Proteins 0.000 claims description 4
- 101000740484 Homo sapiens Aryl hydrocarbon receptor nuclear translocator-like protein 1 Proteins 0.000 claims description 4
- 101000974934 Homo sapiens Cyclic AMP-dependent transcription factor ATF-2 Proteins 0.000 claims description 4
- 101001081582 Homo sapiens DNA-binding protein inhibitor ID-2 Proteins 0.000 claims description 4
- 101000905239 Homo sapiens Heart- and neural crest derivatives-expressed protein 1 Proteins 0.000 claims description 4
- 101000897691 Homo sapiens Helix-loop-helix protein 1 Proteins 0.000 claims description 4
- 101000897700 Homo sapiens Helix-loop-helix protein 2 Proteins 0.000 claims description 4
- 101001046870 Homo sapiens Hypoxia-inducible factor 1-alpha Proteins 0.000 claims description 4
- 101001023043 Homo sapiens Myoblast determination protein 1 Proteins 0.000 claims description 4
- 101000589002 Homo sapiens Myogenin Proteins 0.000 claims description 4
- 101000973177 Homo sapiens Nuclear factor interleukin-3-regulated protein Proteins 0.000 claims description 4
- 101000616761 Homo sapiens Single-minded homolog 2 Proteins 0.000 claims description 4
- 101001041525 Homo sapiens Transcription factor 12 Proteins 0.000 claims description 4
- 101000800563 Homo sapiens Transcription factor 15 Proteins 0.000 claims description 4
- 101000835018 Homo sapiens Transcription factor AP-4 Proteins 0.000 claims description 4
- 101000837845 Homo sapiens Transcription factor E3 Proteins 0.000 claims description 4
- 101000837841 Homo sapiens Transcription factor EB Proteins 0.000 claims description 4
- 101000671637 Homo sapiens Upstream stimulatory factor 1 Proteins 0.000 claims description 4
- 101000671649 Homo sapiens Upstream stimulatory factor 2 Proteins 0.000 claims description 4
- 102100022875 Hypoxia-inducible factor 1-alpha Human genes 0.000 claims description 4
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 4
- 102100035077 Myoblast determination protein 1 Human genes 0.000 claims description 4
- 102100038380 Myogenic factor 5 Human genes 0.000 claims description 4
- 102100038379 Myogenic factor 6 Human genes 0.000 claims description 4
- 102100032970 Myogenin Human genes 0.000 claims description 4
- 102100031701 Nuclear factor erythroid 2-related factor 2 Human genes 0.000 claims description 4
- 102100022163 Nuclear factor interleukin-3-regulated protein Human genes 0.000 claims description 4
- 206010033128 Ovarian cancer Diseases 0.000 claims description 4
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 4
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 4
- 102100021825 Single-minded homolog 2 Human genes 0.000 claims description 4
- 102100026839 Sterol regulatory element-binding protein 1 Human genes 0.000 claims description 4
- 102100026841 Sterol regulatory element-binding protein 2 Human genes 0.000 claims description 4
- 101001023030 Toxoplasma gondii Myosin-D Proteins 0.000 claims description 4
- 102100021123 Transcription factor 12 Human genes 0.000 claims description 4
- 102100033128 Transcription factor 15 Human genes 0.000 claims description 4
- 102100026154 Transcription factor AP-4 Human genes 0.000 claims description 4
- 102100028507 Transcription factor E3 Human genes 0.000 claims description 4
- 102100028502 Transcription factor EB Human genes 0.000 claims description 4
- 102100023118 Transcription factor JunD Human genes 0.000 claims description 4
- 102100040105 Upstream stimulatory factor 1 Human genes 0.000 claims description 4
- 102100040103 Upstream stimulatory factor 2 Human genes 0.000 claims description 4
- 230000000692 anti-sense effect Effects 0.000 claims description 4
- 108010018033 endothelial PAS domain-containing protein 1 Proteins 0.000 claims description 4
- 201000010536 head and neck cancer Diseases 0.000 claims description 4
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 4
- 201000005202 lung cancer Diseases 0.000 claims description 4
- 208000020816 lung neoplasm Diseases 0.000 claims description 4
- 201000000849 skin cancer Diseases 0.000 claims description 4
- 238000012360 testing method Methods 0.000 claims description 4
- YDRYQBCOLJPFFX-REOHCLBHSA-N (2r)-2-amino-3-(1,1,2,2-tetrafluoroethylsulfanyl)propanoic acid Chemical compound OC(=O)[C@@H](N)CSC(F)(F)C(F)F YDRYQBCOLJPFFX-REOHCLBHSA-N 0.000 claims description 3
- 101150059521 AHRR gene Proteins 0.000 claims description 3
- 108010088547 ARNTL Transcription Factors Proteins 0.000 claims description 3
- 102100022142 Achaete-scute homolog 1 Human genes 0.000 claims description 3
- 102100022144 Achaete-scute homolog 2 Human genes 0.000 claims description 3
- 102100022138 Achaete-scute homolog 3 Human genes 0.000 claims description 3
- 102100022137 Achaete-scute homolog 4 Human genes 0.000 claims description 3
- 108010080691 Alcohol O-acetyltransferase Proteins 0.000 claims description 3
- 102100027839 Aryl hydrocarbon receptor nuclear translocator 2 Human genes 0.000 claims description 3
- 102100021661 Aryl hydrocarbon receptor nuclear translocator-like protein 2 Human genes 0.000 claims description 3
- 102100026789 Aryl hydrocarbon receptor repressor Human genes 0.000 claims description 3
- 101150004658 BHLHE22 gene Proteins 0.000 claims description 3
- 101150063122 BHLHE23 gene Proteins 0.000 claims description 3
- 101150050047 BHLHE40 gene Proteins 0.000 claims description 3
- 102100037936 Basic helix-loop-helix domain-containing protein USF3 Human genes 0.000 claims description 3
- 102100031697 Basic helix-loop-helix transcription factor scleraxis Human genes 0.000 claims description 3
- 102100022970 Basic leucine zipper transcriptional factor ATF-like Human genes 0.000 claims description 3
- 102100023006 Basic leucine zipper transcriptional factor ATF-like 2 Human genes 0.000 claims description 3
- 102100023013 Basic leucine zipper transcriptional factor ATF-like 3 Human genes 0.000 claims description 3
- 101150023803 Bhlha15 gene Proteins 0.000 claims description 3
- 101150106705 Bhlhe41 gene Proteins 0.000 claims description 3
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 3
- 101000690445 Caenorhabditis elegans Aryl hydrocarbon receptor nuclear translocator homolog Proteins 0.000 claims description 3
- 102100037403 Carbohydrate-responsive element-binding protein Human genes 0.000 claims description 3
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 3
- 102100026204 Class E basic helix-loop-helix protein 22 Human genes 0.000 claims description 3
- 102100026196 Class E basic helix-loop-helix protein 23 Human genes 0.000 claims description 3
- 102100026191 Class E basic helix-loop-helix protein 40 Human genes 0.000 claims description 3
- 102100026190 Class E basic helix-loop-helix protein 41 Human genes 0.000 claims description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 3
- 102100023580 Cyclic AMP-dependent transcription factor ATF-4 Human genes 0.000 claims description 3
- 102100023582 Cyclic AMP-dependent transcription factor ATF-5 Human genes 0.000 claims description 3
- 102100023583 Cyclic AMP-dependent transcription factor ATF-6 alpha Human genes 0.000 claims description 3
- 102100023581 Cyclic AMP-dependent transcription factor ATF-6 beta Human genes 0.000 claims description 3
- 102100023578 Cyclic AMP-dependent transcription factor ATF-7 Human genes 0.000 claims description 3
- 102100026359 Cyclic AMP-responsive element-binding protein 1 Human genes 0.000 claims description 3
- 102100039297 Cyclic AMP-responsive element-binding protein 3-like protein 1 Human genes 0.000 claims description 3
- 102100039299 Cyclic AMP-responsive element-binding protein 3-like protein 2 Human genes 0.000 claims description 3
- 102100021306 Cyclic AMP-responsive element-binding protein 3-like protein 3 Human genes 0.000 claims description 3
- 102100039436 DNA-binding protein inhibitor ID-3 Human genes 0.000 claims description 3
- 102100039439 DNA-binding protein inhibitor ID-4 Human genes 0.000 claims description 3
- 102100037008 Factor in the germline alpha Human genes 0.000 claims description 3
- 102100031442 Fer3-like protein Human genes 0.000 claims description 3
- 102100028121 Fos-related antigen 2 Human genes 0.000 claims description 3
- 108010081348 HRT1 protein Hairy Proteins 0.000 claims description 3
- 102100021881 Hairy/enhancer-of-split related with YRPW motif protein 1 Human genes 0.000 claims description 3
- 102100039990 Hairy/enhancer-of-split related with YRPW motif protein 2 Human genes 0.000 claims description 3
- 101000901099 Homo sapiens Achaete-scute homolog 1 Proteins 0.000 claims description 3
- 101000901109 Homo sapiens Achaete-scute homolog 2 Proteins 0.000 claims description 3
- 101000901094 Homo sapiens Achaete-scute homolog 3 Proteins 0.000 claims description 3
- 101000901090 Homo sapiens Achaete-scute homolog 4 Proteins 0.000 claims description 3
- 101000768838 Homo sapiens Aryl hydrocarbon receptor nuclear translocator 2 Proteins 0.000 claims description 3
- 101000805924 Homo sapiens Basic helix-loop-helix domain-containing protein USF3 Proteins 0.000 claims description 3
- 101000654285 Homo sapiens Basic helix-loop-helix transcription factor scleraxis Proteins 0.000 claims description 3
- 101000903742 Homo sapiens Basic leucine zipper transcriptional factor ATF-like Proteins 0.000 claims description 3
- 101000903615 Homo sapiens Basic leucine zipper transcriptional factor ATF-like 2 Proteins 0.000 claims description 3
- 101000903609 Homo sapiens Basic leucine zipper transcriptional factor ATF-like 3 Proteins 0.000 claims description 3
- 101000952179 Homo sapiens Carbohydrate-responsive element-binding protein Proteins 0.000 claims description 3
- 101000971300 Homo sapiens Class A basic helix-loop-helix protein 15 Proteins 0.000 claims description 3
- 101000905743 Homo sapiens Cyclic AMP-dependent transcription factor ATF-4 Proteins 0.000 claims description 3
- 101000905746 Homo sapiens Cyclic AMP-dependent transcription factor ATF-5 Proteins 0.000 claims description 3
- 101000905751 Homo sapiens Cyclic AMP-dependent transcription factor ATF-6 alpha Proteins 0.000 claims description 3
- 101000905727 Homo sapiens Cyclic AMP-dependent transcription factor ATF-6 beta Proteins 0.000 claims description 3
- 101000905723 Homo sapiens Cyclic AMP-dependent transcription factor ATF-7 Proteins 0.000 claims description 3
- 101000855516 Homo sapiens Cyclic AMP-responsive element-binding protein 1 Proteins 0.000 claims description 3
- 101000855520 Homo sapiens Cyclic AMP-responsive element-binding protein 3 Proteins 0.000 claims description 3
- 101000745631 Homo sapiens Cyclic AMP-responsive element-binding protein 3-like protein 1 Proteins 0.000 claims description 3
- 101000745624 Homo sapiens Cyclic AMP-responsive element-binding protein 3-like protein 2 Proteins 0.000 claims description 3
- 101000895303 Homo sapiens Cyclic AMP-responsive element-binding protein 3-like protein 3 Proteins 0.000 claims description 3
- 101000726193 Homo sapiens Cyclic AMP-responsive element-binding protein 5 Proteins 0.000 claims description 3
- 101001036287 Homo sapiens DNA-binding protein inhibitor ID-3 Proteins 0.000 claims description 3
- 101001036276 Homo sapiens DNA-binding protein inhibitor ID-4 Proteins 0.000 claims description 3
- 101000878291 Homo sapiens Factor in the germline alpha Proteins 0.000 claims description 3
- 101000848171 Homo sapiens Fanconi anemia group J protein Proteins 0.000 claims description 3
- 101000846731 Homo sapiens Fer3-like protein Proteins 0.000 claims description 3
- 101001059934 Homo sapiens Fos-related antigen 2 Proteins 0.000 claims description 3
- 101000997829 Homo sapiens Glial cell line-derived neurotrophic factor Proteins 0.000 claims description 3
- 101001002170 Homo sapiens Glutamine amidotransferase-like class 1 domain-containing protein 3, mitochondrial Proteins 0.000 claims description 3
- 101001035089 Homo sapiens Hairy/enhancer-of-split related with YRPW motif protein 2 Proteins 0.000 claims description 3
- 101000952181 Homo sapiens MLX-interacting protein Proteins 0.000 claims description 3
- 101000962483 Homo sapiens Max dimerization protein 1 Proteins 0.000 claims description 3
- 101001036585 Homo sapiens Max dimerization protein 3 Proteins 0.000 claims description 3
- 101001036580 Homo sapiens Max dimerization protein 4 Proteins 0.000 claims description 3
- 101000576320 Homo sapiens Max-binding protein MNT Proteins 0.000 claims description 3
- 101001000302 Homo sapiens Max-interacting protein 1 Proteins 0.000 claims description 3
- 101000952182 Homo sapiens Max-like protein X Proteins 0.000 claims description 3
- 101000629402 Homo sapiens Mesoderm posterior protein 1 Proteins 0.000 claims description 3
- 101000629405 Homo sapiens Mesoderm posterior protein 2 Proteins 0.000 claims description 3
- 101001133999 Homo sapiens Mesogenin-1 Proteins 0.000 claims description 3
- 101000958041 Homo sapiens Musculin Proteins 0.000 claims description 3
- 101000958865 Homo sapiens Myogenic factor 5 Proteins 0.000 claims description 3
- 101000958866 Homo sapiens Myogenic factor 6 Proteins 0.000 claims description 3
- 101000582320 Homo sapiens Neurogenic differentiation factor 6 Proteins 0.000 claims description 3
- 101000603763 Homo sapiens Neurogenin-1 Proteins 0.000 claims description 3
- 101000603698 Homo sapiens Neurogenin-2 Proteins 0.000 claims description 3
- 101000603702 Homo sapiens Neurogenin-3 Proteins 0.000 claims description 3
- 101000634538 Homo sapiens Neuronal PAS domain-containing protein 1 Proteins 0.000 claims description 3
- 101000634537 Homo sapiens Neuronal PAS domain-containing protein 2 Proteins 0.000 claims description 3
- 101000634545 Homo sapiens Neuronal PAS domain-containing protein 3 Proteins 0.000 claims description 3
- 101000588303 Homo sapiens Nuclear factor erythroid 2-related factor 3 Proteins 0.000 claims description 3
- 101000602926 Homo sapiens Nuclear receptor coactivator 1 Proteins 0.000 claims description 3
- 101000974356 Homo sapiens Nuclear receptor coactivator 3 Proteins 0.000 claims description 3
- 101001120753 Homo sapiens Oligodendrocyte transcription factor 1 Proteins 0.000 claims description 3
- 101001120819 Homo sapiens Oligodendrocyte transcription factor 3 Proteins 0.000 claims description 3
- 101000738523 Homo sapiens Pancreas transcription factor 1 subunit alpha Proteins 0.000 claims description 3
- 101000585703 Homo sapiens Protein L-Myc Proteins 0.000 claims description 3
- 101000958299 Homo sapiens Protein lyl-1 Proteins 0.000 claims description 3
- 101000584293 Homo sapiens Putative myc-like protein MYCLP1 Proteins 0.000 claims description 3
- 101000703681 Homo sapiens Single-minded homolog 1 Proteins 0.000 claims description 3
- 101000711812 Homo sapiens Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 1 Proteins 0.000 claims description 3
- 101000711810 Homo sapiens Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 2 Proteins 0.000 claims description 3
- 101000629597 Homo sapiens Sterol regulatory element-binding protein 1 Proteins 0.000 claims description 3
- 101000629605 Homo sapiens Sterol regulatory element-binding protein 2 Proteins 0.000 claims description 3
- 101000891113 Homo sapiens T-cell acute lymphocytic leukemia protein 1 Proteins 0.000 claims description 3
- 101000625330 Homo sapiens T-cell acute lymphocytic leukemia protein 2 Proteins 0.000 claims description 3
- 101001067250 Homo sapiens Transcription cofactor HES-6 Proteins 0.000 claims description 3
- 101000800546 Homo sapiens Transcription factor 21 Proteins 0.000 claims description 3
- 101000976959 Homo sapiens Transcription factor 4 Proteins 0.000 claims description 3
- 101000596772 Homo sapiens Transcription factor 7-like 1 Proteins 0.000 claims description 3
- 101000596771 Homo sapiens Transcription factor 7-like 2 Proteins 0.000 claims description 3
- 101000701142 Homo sapiens Transcription factor ATOH1 Proteins 0.000 claims description 3
- 101000701154 Homo sapiens Transcription factor ATOH7 Proteins 0.000 claims description 3
- 101000701302 Homo sapiens Transcription factor ATOH8 Proteins 0.000 claims description 3
- 101000666382 Homo sapiens Transcription factor E2-alpha Proteins 0.000 claims description 3
- 101000837837 Homo sapiens Transcription factor EC Proteins 0.000 claims description 3
- 101000843556 Homo sapiens Transcription factor HES-1 Proteins 0.000 claims description 3
- 101000843572 Homo sapiens Transcription factor HES-2 Proteins 0.000 claims description 3
- 101000843569 Homo sapiens Transcription factor HES-3 Proteins 0.000 claims description 3
- 101000843562 Homo sapiens Transcription factor HES-4 Proteins 0.000 claims description 3
- 101000843449 Homo sapiens Transcription factor HES-5 Proteins 0.000 claims description 3
- 101001067244 Homo sapiens Transcription factor HES-7 Proteins 0.000 claims description 3
- 101001028730 Homo sapiens Transcription factor JunB Proteins 0.000 claims description 3
- 101001050297 Homo sapiens Transcription factor JunD Proteins 0.000 claims description 3
- 101000653542 Homo sapiens Transcription factor-like 5 protein Proteins 0.000 claims description 3
- 101000894871 Homo sapiens Transcription regulator protein BACH1 Proteins 0.000 claims description 3
- 101000904499 Homo sapiens Transcription regulator protein BACH2 Proteins 0.000 claims description 3
- 101000919269 Homo sapiens cAMP-responsive element modulator Proteins 0.000 claims description 3
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 3
- 102100037406 MLX-interacting protein Human genes 0.000 claims description 3
- 102100039185 Max dimerization protein 1 Human genes 0.000 claims description 3
- 102100039513 Max dimerization protein 3 Human genes 0.000 claims description 3
- 102100039515 Max dimerization protein 4 Human genes 0.000 claims description 3
- 102100025169 Max-binding protein MNT Human genes 0.000 claims description 3
- 102100035880 Max-interacting protein 1 Human genes 0.000 claims description 3
- 102100037423 Max-like protein X Human genes 0.000 claims description 3
- 102100026822 Mesoderm posterior protein 1 Human genes 0.000 claims description 3
- 102100026817 Mesoderm posterior protein 2 Human genes 0.000 claims description 3
- 102100034148 Mesogenin-1 Human genes 0.000 claims description 3
- 101100163882 Mus musculus Ascl1 gene Proteins 0.000 claims description 3
- 102100038169 Musculin Human genes 0.000 claims description 3
- 101150079937 NEUROD1 gene Proteins 0.000 claims description 3
- 101150012484 NEUROD4 gene Proteins 0.000 claims description 3
- 101150006690 NEUROD6 gene Proteins 0.000 claims description 3
- 108010071382 NF-E2-Related Factor 2 Proteins 0.000 claims description 3
- 108700020297 NeuroD Proteins 0.000 claims description 3
- 102100032063 Neurogenic differentiation factor 1 Human genes 0.000 claims description 3
- 102100032062 Neurogenic differentiation factor 2 Human genes 0.000 claims description 3
- 102100032061 Neurogenic differentiation factor 4 Human genes 0.000 claims description 3
- 102100038550 Neurogenin-1 Human genes 0.000 claims description 3
- 102100038554 Neurogenin-2 Human genes 0.000 claims description 3
- 102100038553 Neurogenin-3 Human genes 0.000 claims description 3
- 102100029052 Neuronal PAS domain-containing protein 1 Human genes 0.000 claims description 3
- 102100029045 Neuronal PAS domain-containing protein 2 Human genes 0.000 claims description 3
- 102100029051 Neuronal PAS domain-containing protein 3 Human genes 0.000 claims description 3
- 102100031700 Nuclear factor erythroid 2-related factor 3 Human genes 0.000 claims description 3
- 102100037223 Nuclear receptor coactivator 1 Human genes 0.000 claims description 3
- 102100022883 Nuclear receptor coactivator 3 Human genes 0.000 claims description 3
- 102100026073 Oligodendrocyte transcription factor 1 Human genes 0.000 claims description 3
- 102100026056 Oligodendrocyte transcription factor 3 Human genes 0.000 claims description 3
- 102100037878 Pancreas transcription factor 1 subunit alpha Human genes 0.000 claims description 3
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 3
- 208000007913 Pituitary Neoplasms Diseases 0.000 claims description 3
- 206010060862 Prostate cancer Diseases 0.000 claims description 3
- 102100030128 Protein L-Myc Human genes 0.000 claims description 3
- 102100038231 Protein lyl-1 Human genes 0.000 claims description 3
- 102100030632 Putative myc-like protein MYCLP1 Human genes 0.000 claims description 3
- 101100218716 Rattus norvegicus Bhlhb3 gene Proteins 0.000 claims description 3
- 206010038389 Renal cancer Diseases 0.000 claims description 3
- 101100188423 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) OAF1 gene Proteins 0.000 claims description 3
- 101100350434 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ORC3 gene Proteins 0.000 claims description 3
- 101000702553 Schistosoma mansoni Antigen Sm21.7 Proteins 0.000 claims description 3
- 101000714192 Schistosoma mansoni Tegument antigen Proteins 0.000 claims description 3
- 108091081021 Sense strand Proteins 0.000 claims description 3
- 102100031980 Single-minded homolog 1 Human genes 0.000 claims description 3
- 208000032383 Soft tissue cancer Diseases 0.000 claims description 3
- 102100034203 Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 1 Human genes 0.000 claims description 3
- 102100034202 Spermatogenesis- and oogenesis-specific basic helix-loop-helix-containing protein 2 Human genes 0.000 claims description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 3
- 102100025039 T-cell acute lymphocytic leukemia protein 2 Human genes 0.000 claims description 3
- 102100034424 Transcription cofactor HES-6 Human genes 0.000 claims description 3
- 102100033121 Transcription factor 21 Human genes 0.000 claims description 3
- 102100029373 Transcription factor ATOH1 Human genes 0.000 claims description 3
- 102100029372 Transcription factor ATOH7 Human genes 0.000 claims description 3
- 102100030455 Transcription factor ATOH8 Human genes 0.000 claims description 3
- 102100028503 Transcription factor EC Human genes 0.000 claims description 3
- 102100030798 Transcription factor HES-1 Human genes 0.000 claims description 3
- 102100030772 Transcription factor HES-2 Human genes 0.000 claims description 3
- 102100030773 Transcription factor HES-3 Human genes 0.000 claims description 3
- 102100030774 Transcription factor HES-4 Human genes 0.000 claims description 3
- 102100030853 Transcription factor HES-5 Human genes 0.000 claims description 3
- 102100034423 Transcription factor HES-7 Human genes 0.000 claims description 3
- 102100037168 Transcription factor JunB Human genes 0.000 claims description 3
- 102100030647 Transcription factor-like 5 protein Human genes 0.000 claims description 3
- 102100021268 Transcription regulator protein BACH1 Human genes 0.000 claims description 3
- 102100023998 Transcription regulator protein BACH2 Human genes 0.000 claims description 3
- 108010083162 Twist-Related Protein 1 Proteins 0.000 claims description 3
- 108010083176 Twist-Related Protein 2 Proteins 0.000 claims description 3
- 102100030398 Twist-related protein 1 Human genes 0.000 claims description 3
- 102100031720 Twist-related protein 2 Human genes 0.000 claims description 3
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 3
- 208000002495 Uterine Neoplasms Diseases 0.000 claims description 3
- 102100029387 cAMP-responsive element modulator Human genes 0.000 claims description 3
- 201000010881 cervical cancer Diseases 0.000 claims description 3
- 208000029742 colonic neoplasm Diseases 0.000 claims description 3
- 108091000099 cysteine desulfurase Proteins 0.000 claims description 3
- 206010017758 gastric cancer Diseases 0.000 claims description 3
- 201000010982 kidney cancer Diseases 0.000 claims description 3
- 201000007270 liver cancer Diseases 0.000 claims description 3
- 208000014018 liver neoplasm Diseases 0.000 claims description 3
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 3
- 201000001441 melanoma Diseases 0.000 claims description 3
- 210000000653 nervous system Anatomy 0.000 claims description 3
- 201000011682 nervous system cancer Diseases 0.000 claims description 3
- 201000002528 pancreatic cancer Diseases 0.000 claims description 3
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 3
- 201000008261 skin carcinoma Diseases 0.000 claims description 3
- 201000011549 stomach cancer Diseases 0.000 claims description 3
- 206010046766 uterine cancer Diseases 0.000 claims description 3
- 101150022024 MYCN gene Proteins 0.000 claims description 2
- 108700026495 N-Myc Proto-Oncogene Proteins 0.000 claims description 2
- 102100030124 N-myc proto-oncogene protein Human genes 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 201000002511 pituitary cancer Diseases 0.000 claims description 2
- 102100023026 Cyclic AMP-dependent transcription factor ATF-1 Human genes 0.000 claims 1
- 101001030211 Homo sapiens Myc proto-oncogene protein Proteins 0.000 claims 1
- 108700012912 MYCN Proteins 0.000 claims 1
- 102100038313 Transcription factor E2-alpha Human genes 0.000 claims 1
- 230000005764 inhibitory process Effects 0.000 abstract description 10
- 230000009870 specific binding Effects 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 72
- 102000053602 DNA Human genes 0.000 description 49
- 108020004414 DNA Proteins 0.000 description 49
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 46
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 46
- 235000018102 proteins Nutrition 0.000 description 42
- 102000039446 nucleic acids Human genes 0.000 description 39
- 108020004707 nucleic acids Proteins 0.000 description 39
- 230000000694 effects Effects 0.000 description 34
- 229920002477 rna polymer Polymers 0.000 description 33
- 230000003993 interaction Effects 0.000 description 29
- 101150039798 MYC gene Proteins 0.000 description 25
- 239000000203 mixture Substances 0.000 description 25
- 101100239628 Danio rerio myca gene Proteins 0.000 description 24
- 101100459258 Xenopus laevis myc-a gene Proteins 0.000 description 24
- 230000014509 gene expression Effects 0.000 description 22
- 150000001413 amino acids Chemical group 0.000 description 19
- 208000003721 Triple Negative Breast Neoplasms Diseases 0.000 description 17
- 208000022679 triple-negative breast carcinoma Diseases 0.000 description 17
- 238000004088 simulation Methods 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 15
- 235000001014 amino acid Nutrition 0.000 description 15
- 229940024606 amino acid Drugs 0.000 description 15
- 230000008859 change Effects 0.000 description 15
- 238000004458 analytical method Methods 0.000 description 14
- 238000000329 molecular dynamics simulation Methods 0.000 description 14
- 230000001965 increasing effect Effects 0.000 description 12
- 235000000346 sugar Nutrition 0.000 description 12
- 108090000765 processed proteins & peptides Proteins 0.000 description 11
- 238000013518 transcription Methods 0.000 description 11
- 230000035897 transcription Effects 0.000 description 11
- 238000011282 treatment Methods 0.000 description 11
- 230000004663 cell proliferation Effects 0.000 description 10
- 230000000295 complement effect Effects 0.000 description 10
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 10
- 230000006870 function Effects 0.000 description 10
- 239000003446 ligand Substances 0.000 description 10
- 150000003839 salts Chemical class 0.000 description 10
- 125000000217 alkyl group Chemical group 0.000 description 9
- 239000003795 chemical substances by application Substances 0.000 description 9
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 238000013459 approach Methods 0.000 description 8
- 238000004364 calculation method Methods 0.000 description 8
- 125000002091 cationic group Chemical group 0.000 description 8
- 230000022131 cell cycle Effects 0.000 description 8
- 230000001419 dependent effect Effects 0.000 description 8
- 239000000651 prodrug Substances 0.000 description 8
- 229940002612 prodrug Drugs 0.000 description 8
- 108020004459 Small interfering RNA Proteins 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 238000003556 assay Methods 0.000 description 7
- 230000004071 biological effect Effects 0.000 description 7
- 230000010261 cell growth Effects 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- 150000002632 lipids Chemical class 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 108091035707 Consensus sequence Proteins 0.000 description 6
- 230000004568 DNA-binding Effects 0.000 description 6
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 6
- 102000001708 Protein Isoforms Human genes 0.000 description 6
- 108010029485 Protein Isoforms Proteins 0.000 description 6
- 239000002246 antineoplastic agent Substances 0.000 description 6
- 238000000354 decomposition reaction Methods 0.000 description 6
- 238000012217 deletion Methods 0.000 description 6
- 230000037430 deletion Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 108020004999 messenger RNA Proteins 0.000 description 6
- 102000044158 nucleic acid binding protein Human genes 0.000 description 6
- 108700020942 nucleic acid binding protein Proteins 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 229940124597 therapeutic agent Drugs 0.000 description 6
- UHDGCWIWMRVCDJ-UHFFFAOYSA-N 1-beta-D-Xylofuranosyl-NH-Cytosine Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 UHDGCWIWMRVCDJ-UHFFFAOYSA-N 0.000 description 5
- 108010027344 Basic Helix-Loop-Helix Transcription Factors Proteins 0.000 description 5
- 102000018720 Basic Helix-Loop-Helix Transcription Factors Human genes 0.000 description 5
- UHDGCWIWMRVCDJ-PSQAKQOGSA-N Cytidine Natural products O=C1N=C(N)C=CN1[C@@H]1[C@@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-PSQAKQOGSA-N 0.000 description 5
- 102100028092 Homeobox protein Nkx-3.1 Human genes 0.000 description 5
- 101000578249 Homo sapiens Homeobox protein Nkx-3.1 Proteins 0.000 description 5
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- UHDGCWIWMRVCDJ-ZAKLUEHWSA-N cytidine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O1 UHDGCWIWMRVCDJ-ZAKLUEHWSA-N 0.000 description 5
- 229940104302 cytosine Drugs 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 5
- 229920000642 polymer Polymers 0.000 description 5
- 238000007614 solvation Methods 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- 238000013519 translation Methods 0.000 description 5
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 4
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 description 4
- 125000003342 alkenyl group Chemical group 0.000 description 4
- 125000000304 alkynyl group Chemical group 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 125000004432 carbon atom Chemical group C* 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 239000000539 dimer Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000010195 expression analysis Methods 0.000 description 4
- 238000000684 flow cytometry Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 230000002452 interceptive effect Effects 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 239000010452 phosphate Chemical group 0.000 description 4
- 125000004437 phosphorous atom Chemical group 0.000 description 4
- 229910052698 phosphorus Inorganic materials 0.000 description 4
- 230000004962 physiological condition Effects 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 4
- 238000001890 transfection Methods 0.000 description 4
- 230000004614 tumor growth Effects 0.000 description 4
- 229930024421 Adenine Natural products 0.000 description 3
- 108091023037 Aptamer Proteins 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- 102100037676 CCAAT/enhancer-binding protein zeta Human genes 0.000 description 3
- 108050006400 Cyclin Proteins 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102100023374 Forkhead box protein M1 Human genes 0.000 description 3
- 102100033295 Glial cell line-derived neurotrophic factor Human genes 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 102100022819 MHC class II regulatory factor RFX1 Human genes 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- 108091057508 Myc family Proteins 0.000 description 3
- 102000008125 NF-kappa B p52 Subunit Human genes 0.000 description 3
- 108010074852 NF-kappa B p52 Subunit Proteins 0.000 description 3
- 108091005461 Nucleic proteins Proteins 0.000 description 3
- 102100036691 Proliferating cell nuclear antigen Human genes 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 108091027967 Small hairpin RNA Proteins 0.000 description 3
- 102100035097 Transcription factor 7-like 1 Human genes 0.000 description 3
- 102100021783 Transcription factor E2F4 Human genes 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 229940059260 amidate Drugs 0.000 description 3
- 150000001408 amides Chemical group 0.000 description 3
- 238000012230 antisense oligonucleotides Methods 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- MVCRZALXJBDOKF-JPZHCBQBSA-N beta-hydroxywybutosine 5'-monophosphate Chemical compound C1=NC=2C(=O)N3C(CC(O)[C@H](NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O MVCRZALXJBDOKF-JPZHCBQBSA-N 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 230000008512 biological response Effects 0.000 description 3
- 238000004422 calculation algorithm Methods 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 230000036755 cellular response Effects 0.000 description 3
- 230000005754 cellular signaling Effects 0.000 description 3
- 230000004700 cellular uptake Effects 0.000 description 3
- 235000012000 cholesterol Nutrition 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 230000009881 electrostatic interaction Effects 0.000 description 3
- 239000003623 enhancer Substances 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 239000000833 heterodimer Substances 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 238000003064 k means clustering Methods 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 235000018977 lysine Nutrition 0.000 description 3
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 229920000962 poly(amidoamine) Polymers 0.000 description 3
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000003584 silencer Effects 0.000 description 3
- 239000004055 small Interfering RNA Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 150000008163 sugars Chemical class 0.000 description 3
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 229940035893 uracil Drugs 0.000 description 3
- 238000001262 western blot Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- HXVKEKIORVUWDR-FDDDBJFASA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-(methylaminomethyl)-2-sulfanylidenepyrimidin-4-one Chemical compound S=C1NC(=O)C(CNC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 HXVKEKIORVUWDR-FDDDBJFASA-N 0.000 description 2
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- ICSNLGPSRYBMBD-UHFFFAOYSA-N 2-aminopyridine Chemical compound NC1=CC=CC=N1 ICSNLGPSRYBMBD-UHFFFAOYSA-N 0.000 description 2
- VZQXUWKZDSEQRR-SDBHATRESA-N 2-methylthio-N(6)-(Delta(2)-isopentenyl)adenosine Chemical compound C12=NC(SC)=NC(NCC=C(C)C)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VZQXUWKZDSEQRR-SDBHATRESA-N 0.000 description 2
- 125000003903 2-propenyl group Chemical group [H]C([*])([H])C([H])=C([H])[H] 0.000 description 2
- BINGDNLMMYSZFR-QYVSTXNMSA-N 3-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-6,7-dimethyl-5h-imidazo[1,2-a]purin-9-one Chemical compound C1=NC=2C(=O)N3C(C)=C(C)N=C3NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O BINGDNLMMYSZFR-QYVSTXNMSA-N 0.000 description 2
- QUZQVVNSDQCAOL-WOUKDFQISA-N 4-demethylwyosine Chemical compound N1C(C)=CN(C(C=2N=C3)=O)C1=NC=2N3[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O QUZQVVNSDQCAOL-WOUKDFQISA-N 0.000 description 2
- OVONXEQGWXGFJD-UHFFFAOYSA-N 4-sulfanylidene-1h-pyrimidin-2-one Chemical compound SC=1C=CNC(=O)N=1 OVONXEQGWXGFJD-UHFFFAOYSA-N 0.000 description 2
- ZLOIGESWDJYCTF-XVFCMESISA-N 4-thiouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=S)C=C1 ZLOIGESWDJYCTF-XVFCMESISA-N 0.000 description 2
- VSCNRXVDHRNJOA-PNHWDRBUSA-N 5-(carboxymethylaminomethyl)uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(CNCC(O)=O)=C1 VSCNRXVDHRNJOA-PNHWDRBUSA-N 0.000 description 2
- RYVNIFSIEDRLSJ-UHFFFAOYSA-N 5-(hydroxymethyl)cytosine Chemical compound NC=1NC(=O)N=CC=1CO RYVNIFSIEDRLSJ-UHFFFAOYSA-N 0.000 description 2
- VKLFQTYNHLDMDP-PNHWDRBUSA-N 5-carboxymethylaminomethyl-2-thiouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=S)NC(=O)C(CNCC(O)=O)=C1 VKLFQTYNHLDMDP-PNHWDRBUSA-N 0.000 description 2
- QXDXBKZJFLRLCM-UAKXSSHOSA-N 5-hydroxyuridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(O)=C1 QXDXBKZJFLRLCM-UAKXSSHOSA-N 0.000 description 2
- HLZXTFWTDIBXDF-PNHWDRBUSA-N 5-methoxycarbonylmethyl-2-thiouridine Chemical compound S=C1NC(=O)C(CC(=O)OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 HLZXTFWTDIBXDF-PNHWDRBUSA-N 0.000 description 2
- YIZYCHKPHCPKHZ-PNHWDRBUSA-N 5-methoxycarbonylmethyluridine Chemical compound O=C1NC(=O)C(CC(=O)OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 YIZYCHKPHCPKHZ-PNHWDRBUSA-N 0.000 description 2
- SNNBPMAXGYBMHM-JXOAFFINSA-N 5-methyl-2-thiouridine Chemical compound S=C1NC(=O)C(C)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 SNNBPMAXGYBMHM-JXOAFFINSA-N 0.000 description 2
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 2
- HCGHYQLFMPXSDU-UHFFFAOYSA-N 7-methyladenine Chemical compound C1=NC(N)=C2N(C)C=NC2=N1 HCGHYQLFMPXSDU-UHFFFAOYSA-N 0.000 description 2
- UJOBWOGCFQCDNV-UHFFFAOYSA-N 9H-carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 description 2
- MSSXOMSJDRHRMC-UHFFFAOYSA-N 9H-purine-2,6-diamine Chemical compound NC1=NC(N)=C2NC=NC2=N1 MSSXOMSJDRHRMC-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 102100036464 Activated RNA polymerase II transcriptional coactivator p15 Human genes 0.000 description 2
- 102100033658 Alpha-globin transcription factor CP2 Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 201000004569 Blindness Diseases 0.000 description 2
- 102100028226 COUP transcription factor 2 Human genes 0.000 description 2
- 101000850966 Cavia porcellus Eosinophil granule major basic protein 1 Proteins 0.000 description 2
- 108010068106 Cyclin T Proteins 0.000 description 2
- 206010061818 Disease progression Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108700011215 E-Box Elements Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 241000283086 Equidae Species 0.000 description 2
- 102100031690 Erythroid transcription factor Human genes 0.000 description 2
- 101150031329 Ets1 gene Proteins 0.000 description 2
- 102100035134 Forkhead box protein J2 Human genes 0.000 description 2
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 2
- 102100035184 General transcription and DNA repair factor IIH helicase subunit XPD Human genes 0.000 description 2
- 102100033840 General transcription factor IIF subunit 1 Human genes 0.000 description 2
- 102100032863 General transcription factor IIH subunit 3 Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 102100032606 Heat shock factor protein 1 Human genes 0.000 description 2
- 102100027489 Helicase-like transcription factor Human genes 0.000 description 2
- 108010020382 Hepatocyte Nuclear Factor 1-alpha Proteins 0.000 description 2
- 102100022057 Hepatocyte nuclear factor 1-alpha Human genes 0.000 description 2
- 108090000246 Histone acetyltransferases Proteins 0.000 description 2
- 102000003893 Histone acetyltransferases Human genes 0.000 description 2
- 102100030339 Homeobox protein Hox-A10 Human genes 0.000 description 2
- 102100025110 Homeobox protein Hox-A5 Human genes 0.000 description 2
- 101000882335 Homo sapiens Alpha-enolase Proteins 0.000 description 2
- 101000907578 Homo sapiens Forkhead box protein M1 Proteins 0.000 description 2
- 101000666405 Homo sapiens General transcription factor IIH subunit 1 Proteins 0.000 description 2
- 101000655398 Homo sapiens General transcription factor IIH subunit 2 Proteins 0.000 description 2
- 101000655391 Homo sapiens General transcription factor IIH subunit 3 Proteins 0.000 description 2
- 101000655406 Homo sapiens General transcription factor IIH subunit 4 Proteins 0.000 description 2
- 101000655402 Homo sapiens General transcription factor IIH subunit 5 Proteins 0.000 description 2
- 101000867525 Homo sapiens Heat shock factor protein 1 Proteins 0.000 description 2
- 101001081105 Homo sapiens Helicase-like transcription factor Proteins 0.000 description 2
- 101001083164 Homo sapiens Homeobox protein Hox-A10 Proteins 0.000 description 2
- 101001077568 Homo sapiens Homeobox protein Hox-A5 Proteins 0.000 description 2
- 101000840577 Homo sapiens Insulin-like growth factor-binding protein 7 Proteins 0.000 description 2
- 101000756759 Homo sapiens MHC class II regulatory factor RFX1 Proteins 0.000 description 2
- 101000614841 Homo sapiens Myocyte-specific enhancer factor 2A Proteins 0.000 description 2
- 101000756346 Homo sapiens RE1-silencing transcription factor Proteins 0.000 description 2
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 2
- 101000895882 Homo sapiens Transcription factor E2F4 Proteins 0.000 description 2
- 101000879604 Homo sapiens Transcription factor E4F1 Proteins 0.000 description 2
- 101000723923 Homo sapiens Transcription factor HIVEP2 Proteins 0.000 description 2
- 101000785626 Homo sapiens Zinc finger E-box-binding homeobox 1 Proteins 0.000 description 2
- 101000723920 Homo sapiens Zinc finger protein 40 Proteins 0.000 description 2
- 229930010555 Inosine Natural products 0.000 description 2
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 2
- 102100029228 Insulin-like growth factor-binding protein 7 Human genes 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 239000012098 Lipofectamine RNAiMAX Substances 0.000 description 2
- 101100445103 Mus musculus Emx2 gene Proteins 0.000 description 2
- 101710150912 Myc protein Proteins 0.000 description 2
- 102100021148 Myocyte-specific enhancer factor 2A Human genes 0.000 description 2
- MBLBDJOUHNCFQT-UHFFFAOYSA-N N-acetyl-D-galactosamine Natural products CC(=O)NC(C=O)C(O)C(O)C(O)CO MBLBDJOUHNCFQT-UHFFFAOYSA-N 0.000 description 2
- 108010057466 NF-kappa B Proteins 0.000 description 2
- 108010018525 NFATC Transcription Factors Proteins 0.000 description 2
- 102000002673 NFATC Transcription Factors Human genes 0.000 description 2
- 102100023050 Nuclear factor NF-kappa-B p105 subunit Human genes 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 108700020796 Oncogene Proteins 0.000 description 2
- 102000007354 PAX6 Transcription Factor Human genes 0.000 description 2
- 108010032788 PAX6 Transcription Factor Proteins 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 102100022940 RE1-silencing transcription factor Human genes 0.000 description 2
- 238000003559 RNA-seq method Methods 0.000 description 2
- 108091030071 RNAI Proteins 0.000 description 2
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 2
- 108020004682 Single-Stranded DNA Proteins 0.000 description 2
- 208000000453 Skin Neoplasms Diseases 0.000 description 2
- 108010074436 Sterol Regulatory Element Binding Protein 1 Proteins 0.000 description 2
- 102100040296 TATA-box-binding protein Human genes 0.000 description 2
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 2
- 239000004473 Threonine Substances 0.000 description 2
- 108091046915 Threose nucleic acid Proteins 0.000 description 2
- 108010048992 Transcription Factor 4 Proteins 0.000 description 2
- 102100024026 Transcription factor E2F1 Human genes 0.000 description 2
- 102100037331 Transcription factor E4F1 Human genes 0.000 description 2
- 102100028438 Transcription factor HIVEP2 Human genes 0.000 description 2
- 102100028604 Transcription initiation factor IIA subunit 2 Human genes 0.000 description 2
- 102100035222 Transcription initiation factor TFIID subunit 1 Human genes 0.000 description 2
- 102100035559 Transcriptional activator GLI3 Human genes 0.000 description 2
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 108010067390 Viral Proteins Proteins 0.000 description 2
- 108010016200 Zinc Finger Protein GLI1 Proteins 0.000 description 2
- 102100026457 Zinc finger E-box-binding homeobox 1 Human genes 0.000 description 2
- 102100028440 Zinc finger protein 40 Human genes 0.000 description 2
- 102100035535 Zinc finger protein GLI1 Human genes 0.000 description 2
- DZBUGLKDJFMEHC-UHFFFAOYSA-N acridine Chemical compound C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 2
- 239000013566 allergen Substances 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 206010002026 amyotrophic lateral sclerosis Diseases 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 239000012298 atmosphere Substances 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 230000025084 cell cycle arrest Effects 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 235000010980 cellulose Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 239000008139 complexing agent Substances 0.000 description 2
- 238000004590 computer program Methods 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- FPUGCISOLXNPPC-IOSLPCCCSA-N cordysinin B Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(N)=C2N=C1 FPUGCISOLXNPPC-IOSLPCCCSA-N 0.000 description 2
- 125000000753 cycloalkyl group Chemical group 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000000326 densiometry Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 2
- 230000005750 disease progression Effects 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 238000009510 drug design Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000005421 electrostatic potential Methods 0.000 description 2
- UVCJGUGAGLDPAA-UHFFFAOYSA-N ensulizole Chemical compound N1C2=CC(S(=O)(=O)O)=CC=C2N=C1C1=CC=CC=C1 UVCJGUGAGLDPAA-UHFFFAOYSA-N 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 101150051296 foxj2 gene Proteins 0.000 description 2
- 125000001475 halogen functional group Chemical group 0.000 description 2
- 230000002489 hematologic effect Effects 0.000 description 2
- 125000000623 heterocyclic group Chemical group 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000126 in silico method Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 229960003786 inosine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- XOTXNXXJZCFUOA-UGKPPGOTSA-N methyl 2-[1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2,4-dioxopyrimidin-5-yl]acetate Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(CC(=O)OC)=C1 XOTXNXXJZCFUOA-UGKPPGOTSA-N 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 2
- 108700024542 myc Genes Proteins 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- RDOWQLZANAYVLL-UHFFFAOYSA-N phenanthridine Chemical compound C1=CC=C2C3=CC=CC=C3C=NC2=C1 RDOWQLZANAYVLL-UHFFFAOYSA-N 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- PTMHPRAIXMAOOB-UHFFFAOYSA-N phosphoramidic acid Chemical compound NP(O)(O)=O PTMHPRAIXMAOOB-UHFFFAOYSA-N 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 229920009537 polybutylene succinate adipate Polymers 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 230000001124 posttranscriptional effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000001959 radiotherapy Methods 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 229910001415 sodium ion Inorganic materials 0.000 description 2
- 108010004034 stable plasma protein solution Proteins 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 239000003826 tablet Substances 0.000 description 2
- 108010067247 tacrolimus binding protein 4 Proteins 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 108010014677 transcription factor TFIIE Proteins 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 1
- GRYSXUXXBDSYRT-WOUKDFQISA-N (2r,3r,4r,5r)-2-(hydroxymethyl)-4-methoxy-5-[6-(methylamino)purin-9-yl]oxolan-3-ol Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1OC GRYSXUXXBDSYRT-WOUKDFQISA-N 0.000 description 1
- DJONVIMMDYQLKR-WOUKDFQISA-N (2r,3r,4r,5r)-2-(hydroxymethyl)-5-(6-imino-1-methylpurin-9-yl)-4-methoxyoxolan-3-ol Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CN(C)C2=N)=C2N=C1 DJONVIMMDYQLKR-WOUKDFQISA-N 0.000 description 1
- YIMATHOGWXZHFX-WCTZXXKLSA-N (2r,3r,4r,5r)-5-(hydroxymethyl)-3-(2-methoxyethoxy)oxolane-2,4-diol Chemical compound COCCO[C@H]1[C@H](O)O[C@H](CO)[C@H]1O YIMATHOGWXZHFX-WCTZXXKLSA-N 0.000 description 1
- MXYRZDAGKTVQIL-IOSLPCCCSA-N (2r,3r,4s,5r)-2-(6-aminopurin-9-yl)-5-(hydroxymethyl)-2-methyloxolane-3,4-diol Chemical compound C1=NC2=C(N)N=CN=C2N1[C@]1(C)O[C@H](CO)[C@@H](O)[C@H]1O MXYRZDAGKTVQIL-IOSLPCCCSA-N 0.000 description 1
- PHFMCMDFWSZKGD-IOSLPCCCSA-N (2r,3s,4r,5r)-2-(hydroxymethyl)-5-[6-(methylamino)-2-methylsulfanylpurin-9-yl]oxolane-3,4-diol Chemical compound C1=NC=2C(NC)=NC(SC)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O PHFMCMDFWSZKGD-IOSLPCCCSA-N 0.000 description 1
- MYUOTPIQBPUQQU-CKTDUXNWSA-N (2s,3r)-2-amino-n-[[9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-methylsulfanylpurin-6-yl]carbamoyl]-3-hydroxybutanamide Chemical compound C12=NC(SC)=NC(NC(=O)NC(=O)[C@@H](N)[C@@H](C)O)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O MYUOTPIQBPUQQU-CKTDUXNWSA-N 0.000 description 1
- GPTUGCGYEMEAOC-IBZYUGMLSA-N (2s,3r)-2-amino-n-[[9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]-methylcarbamoyl]-3-hydroxybutanamide Chemical compound C1=NC=2C(N(C)C(=O)NC(=O)[C@@H](N)[C@H](O)C)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O GPTUGCGYEMEAOC-IBZYUGMLSA-N 0.000 description 1
- JZSSTKLEXRQFEA-HEIFUQTGSA-N (2s,3r,4s,5r)-2-(6-aminopurin-9-yl)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-carboxamide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@]1(C(=O)N)O[C@H](CO)[C@@H](O)[C@H]1O JZSSTKLEXRQFEA-HEIFUQTGSA-N 0.000 description 1
- XBBQCOKPWNZHFX-TYASJMOZSA-N (3r,4s,5r)-2-[(2r,3r,4r,5r)-2-(6-aminopurin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy-5-(hydroxymethyl)oxolane-3,4-diol Chemical compound O([C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C=2N=CN=C(C=2N=C1)N)C1O[C@H](CO)[C@@H](O)[C@H]1O XBBQCOKPWNZHFX-TYASJMOZSA-N 0.000 description 1
- 125000000008 (C1-C10) alkyl group Chemical group 0.000 description 1
- UTZAFOQPCXRRFF-RKBILKOESA-N (beta-D-glucosyl)-O-mycofactocinone Chemical compound CC1(C(NC(=O)C1=O)CC2=CC=C(C=C2)O[C@H]3[C@@H]([C@H]([C@@H]([C@H](O3)CO)O)O)O)C UTZAFOQPCXRRFF-RKBILKOESA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- UFSCXDAOCAIFOG-UHFFFAOYSA-N 1,10-dihydropyrimido[5,4-b][1,4]benzothiazin-2-one Chemical compound S1C2=CC=CC=C2N=C2C1=CNC(=O)N2 UFSCXDAOCAIFOG-UHFFFAOYSA-N 0.000 description 1
- PTFYZDMJTFMPQW-UHFFFAOYSA-N 1,10-dihydropyrimido[5,4-b][1,4]benzoxazin-2-one Chemical compound O1C2=CC=CC=C2N=C2C1=CNC(=O)N2 PTFYZDMJTFMPQW-UHFFFAOYSA-N 0.000 description 1
- FYADHXFMURLYQI-UHFFFAOYSA-N 1,2,4-triazine Chemical class C1=CN=NC=N1 FYADHXFMURLYQI-UHFFFAOYSA-N 0.000 description 1
- OTFGHFBGGZEXEU-PEBGCTIMSA-N 1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-3-methylpyrimidine-2,4-dione Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N(C)C(=O)C=C1 OTFGHFBGGZEXEU-PEBGCTIMSA-N 0.000 description 1
- XIJAZGMFHRTBFY-FDDDBJFASA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-$l^{1}-selanyl-5-(methylaminomethyl)pyrimidin-4-one Chemical compound [Se]C1=NC(=O)C(CNC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 XIJAZGMFHRTBFY-FDDDBJFASA-N 0.000 description 1
- UTQUILVPBZEHTK-ZOQUXTDFSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3-methylpyrimidine-2,4-dione Chemical compound O=C1N(C)C(=O)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 UTQUILVPBZEHTK-ZOQUXTDFSA-N 0.000 description 1
- BTFXIEGOSDSOGN-KWCDMSRLSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-5-methyl-1,3-diazinane-2,4-dione Chemical compound O=C1NC(=O)C(C)CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 BTFXIEGOSDSOGN-KWCDMSRLSA-N 0.000 description 1
- BNXGRQLXOMSOMV-UHFFFAOYSA-N 1-[4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-4-(methylamino)pyrimidin-2-one Chemical compound O=C1N=C(NC)C=CN1C1C(OC)C(O)C(CO)O1 BNXGRQLXOMSOMV-UHFFFAOYSA-N 0.000 description 1
- GFYLSDSUCHVORB-IOSLPCCCSA-N 1-methyladenosine Chemical compound C1=NC=2C(=N)N(C)C=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O GFYLSDSUCHVORB-IOSLPCCCSA-N 0.000 description 1
- UTAIYTHAJQNQDW-KQYNXXCUSA-N 1-methylguanosine Chemical compound C1=NC=2C(=O)N(C)C(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O UTAIYTHAJQNQDW-KQYNXXCUSA-N 0.000 description 1
- WJNGQIYEQLPJMN-IOSLPCCCSA-N 1-methylinosine Chemical compound C1=NC=2C(=O)N(C)C=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O WJNGQIYEQLPJMN-IOSLPCCCSA-N 0.000 description 1
- WJFKNYWRSNBZNX-UHFFFAOYSA-N 10H-phenothiazine Chemical compound C1=CC=C2NC3=CC=CC=C3SC2=C1 WJFKNYWRSNBZNX-UHFFFAOYSA-N 0.000 description 1
- TZMSYXZUNZXBOL-UHFFFAOYSA-N 10H-phenoxazine Chemical compound C1=CC=C2NC3=CC=CC=C3OC2=C1 TZMSYXZUNZXBOL-UHFFFAOYSA-N 0.000 description 1
- UHUHBFMZVCOEOV-UHFFFAOYSA-N 1h-imidazo[4,5-c]pyridin-4-amine Chemical compound NC1=NC=CC2=C1N=CN2 UHUHBFMZVCOEOV-UHFFFAOYSA-N 0.000 description 1
- FPUGCISOLXNPPC-UHFFFAOYSA-N 2'-O-Methyladenosine Natural products COC1C(O)C(CO)OC1N1C2=NC=NC(N)=C2N=C1 FPUGCISOLXNPPC-UHFFFAOYSA-N 0.000 description 1
- RFCQJGFZUQFYRF-UHFFFAOYSA-N 2'-O-Methylcytidine Natural products COC1C(O)C(CO)OC1N1C(=O)N=C(N)C=C1 RFCQJGFZUQFYRF-UHFFFAOYSA-N 0.000 description 1
- OVYNGSFVYRPRCG-UHFFFAOYSA-N 2'-O-Methylguanosine Natural products COC1C(O)C(CO)OC1N1C(NC(N)=NC2=O)=C2N=C1 OVYNGSFVYRPRCG-UHFFFAOYSA-N 0.000 description 1
- SXUXMRMBWZCMEN-UHFFFAOYSA-N 2'-O-methyl uridine Natural products COC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 SXUXMRMBWZCMEN-UHFFFAOYSA-N 0.000 description 1
- RFCQJGFZUQFYRF-ZOQUXTDFSA-N 2'-O-methylcytidine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C=C1 RFCQJGFZUQFYRF-ZOQUXTDFSA-N 0.000 description 1
- OVYNGSFVYRPRCG-KQYNXXCUSA-N 2'-O-methylguanosine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=C(N)NC2=O)=C2N=C1 OVYNGSFVYRPRCG-KQYNXXCUSA-N 0.000 description 1
- HPHXOIULGYVAKW-IOSLPCCCSA-N 2'-O-methylinosine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC2=O)=C2N=C1 HPHXOIULGYVAKW-IOSLPCCCSA-N 0.000 description 1
- HPHXOIULGYVAKW-UHFFFAOYSA-N 2'-O-methylinosine Natural products COC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 HPHXOIULGYVAKW-UHFFFAOYSA-N 0.000 description 1
- RSMRWWHFJMENJH-LQDDAWAPSA-M 2,3-bis[[(z)-octadec-9-enoyl]oxy]propyl-trimethylazanium;methyl sulfate Chemical compound COS([O-])(=O)=O.CCCCCCCC\C=C/CCCCCCCC(=O)OCC(C[N+](C)(C)C)OC(=O)CCCCCCC\C=C/CCCCCCCC RSMRWWHFJMENJH-LQDDAWAPSA-M 0.000 description 1
- VEPOHXYIFQMVHW-XOZOLZJESA-N 2,3-dihydroxybutanedioic acid (2S,3S)-3,4-dimethyl-2-phenylmorpholine Chemical compound OC(C(O)C(O)=O)C(O)=O.C[C@H]1[C@@H](OCCN1C)c1ccccc1 VEPOHXYIFQMVHW-XOZOLZJESA-N 0.000 description 1
- QSHACTSJHMKXTE-UHFFFAOYSA-N 2-(2-aminopropyl)-7h-purin-6-amine Chemical compound CC(N)CC1=NC(N)=C2NC=NC2=N1 QSHACTSJHMKXTE-UHFFFAOYSA-N 0.000 description 1
- YUCFXTKBZFABID-WOUKDFQISA-N 2-(dimethylamino)-9-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-3h-purin-6-one Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(NC(=NC2=O)N(C)C)=C2N=C1 YUCFXTKBZFABID-WOUKDFQISA-N 0.000 description 1
- IQZWKGWOBPJWMX-UHFFFAOYSA-N 2-Methyladenosine Natural products C12=NC(C)=NC(N)=C2N=CN1C1OC(CO)C(O)C1O IQZWKGWOBPJWMX-UHFFFAOYSA-N 0.000 description 1
- VHXUHQJRMXUOST-PNHWDRBUSA-N 2-[1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2,4-dioxopyrimidin-5-yl]acetamide Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(CC(N)=O)=C1 VHXUHQJRMXUOST-PNHWDRBUSA-N 0.000 description 1
- AKWUNZFZIXEOPV-UHFFFAOYSA-N 2-[4-[[3-[7-chloro-1-(oxan-4-ylmethyl)indol-3-yl]-1,2,4-oxadiazol-5-yl]methyl]piperazin-1-yl]acetamide Chemical compound C1CN(CC(=O)N)CCN1CC1=NC(C=2C3=CC=CC(Cl)=C3N(CC3CCOCC3)C=2)=NO1 AKWUNZFZIXEOPV-UHFFFAOYSA-N 0.000 description 1
- SFFCQAIBJUCFJK-UGKPPGOTSA-N 2-[[1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2,4-dioxopyrimidin-5-yl]methylamino]acetic acid Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(CNCC(O)=O)=C1 SFFCQAIBJUCFJK-UGKPPGOTSA-N 0.000 description 1
- VJKJOPUEUOTEBX-TURQNECASA-N 2-[[1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2,4-dioxopyrimidin-5-yl]methylamino]ethanesulfonic acid Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(CNCCS(O)(=O)=O)=C1 VJKJOPUEUOTEBX-TURQNECASA-N 0.000 description 1
- OSBLTNPMIGYQGY-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;boric acid Chemical compound OB(O)O.OCC(N)(CO)CO.OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O OSBLTNPMIGYQGY-UHFFFAOYSA-N 0.000 description 1
- SOEYIPCQNRSIAV-IOSLPCCCSA-N 2-amino-5-(aminomethyl)-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=2NC(N)=NC(=O)C=2C(CN)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O SOEYIPCQNRSIAV-IOSLPCCCSA-N 0.000 description 1
- JRYMOPZHXMVHTA-DAGMQNCNSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1h-pyrrolo[2,3-d]pyrimidin-4-one Chemical compound C1=CC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JRYMOPZHXMVHTA-DAGMQNCNSA-N 0.000 description 1
- BIRQNXWAXWLATA-IOSLPCCCSA-N 2-amino-7-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4-oxo-1h-pyrrolo[2,3-d]pyrimidine-5-carbonitrile Chemical compound C1=C(C#N)C=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O BIRQNXWAXWLATA-IOSLPCCCSA-N 0.000 description 1
- NTYZLKZZBRSAPT-DBINCYRJSA-N 2-amino-9-[(2r,3r,4r,5r)-3-[(3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]oxy-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-3h-purin-6-one Chemical compound O([C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C=NC=2C(=O)N=C(NC=21)N)C1O[C@H](CO)[C@@H](O)[C@H]1O NTYZLKZZBRSAPT-DBINCYRJSA-N 0.000 description 1
- JLYURAYAEKVGQJ-IOSLPCCCSA-N 2-amino-9-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-1-methylpurin-6-one Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=C(N)N(C)C2=O)=C2N=C1 JLYURAYAEKVGQJ-IOSLPCCCSA-N 0.000 description 1
- PBFLIOAJBULBHI-JJNLEZRASA-N 2-amino-n-[[9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]carbamoyl]acetamide Chemical compound C1=NC=2C(NC(=O)NC(=O)CN)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O PBFLIOAJBULBHI-JJNLEZRASA-N 0.000 description 1
- WKMPTBDYDNUJLF-UHFFFAOYSA-N 2-fluoroadenine Chemical compound NC1=NC(F)=NC2=C1N=CN2 WKMPTBDYDNUJLF-UHFFFAOYSA-N 0.000 description 1
- 125000004200 2-methoxyethyl group Chemical group [H]C([H])([H])OC([H])([H])C([H])([H])* 0.000 description 1
- VWSLLSXLURJCDF-UHFFFAOYSA-N 2-methyl-4,5-dihydro-1h-imidazole Chemical compound CC1=NCCN1 VWSLLSXLURJCDF-UHFFFAOYSA-N 0.000 description 1
- IQZWKGWOBPJWMX-IOSLPCCCSA-N 2-methyladenosine Chemical compound C12=NC(C)=NC(N)=C2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O IQZWKGWOBPJWMX-IOSLPCCCSA-N 0.000 description 1
- QEWSGVMSLPHELX-UHFFFAOYSA-N 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine Chemical compound C12=NC(SC)=NC(NCC=C(C)CO)=C2N=CN1C1OC(CO)C(O)C1O QEWSGVMSLPHELX-UHFFFAOYSA-N 0.000 description 1
- RHFUOMFWUGWKKO-XVFCMESISA-N 2-thiocytidine Chemical compound S=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RHFUOMFWUGWKKO-XVFCMESISA-N 0.000 description 1
- OALHHIHQOFIMEF-UHFFFAOYSA-N 3',6'-dihydroxy-2',4',5',7'-tetraiodo-3h-spiro[2-benzofuran-1,9'-xanthene]-3-one Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 OALHHIHQOFIMEF-UHFFFAOYSA-N 0.000 description 1
- YXNIEZJFCGTDKV-JANFQQFMSA-N 3-(3-amino-3-carboxypropyl)uridine Chemical compound O=C1N(CCC(N)C(O)=O)C(=O)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 YXNIEZJFCGTDKV-JANFQQFMSA-N 0.000 description 1
- RDPUKVRQKWBSPK-UHFFFAOYSA-N 3-Methylcytidine Natural products O=C1N(C)C(=N)C=CN1C1C(O)C(O)C(CO)O1 RDPUKVRQKWBSPK-UHFFFAOYSA-N 0.000 description 1
- UTQUILVPBZEHTK-UHFFFAOYSA-N 3-Methyluridine Natural products O=C1N(C)C(=O)C=CN1C1C(O)C(O)C(CO)O1 UTQUILVPBZEHTK-UHFFFAOYSA-N 0.000 description 1
- HOEIPINIBKBXTJ-IDTAVKCVSA-N 3-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4,6,7-trimethylimidazo[1,2-a]purin-9-one Chemical compound C1=NC=2C(=O)N3C(C)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O HOEIPINIBKBXTJ-IDTAVKCVSA-N 0.000 description 1
- RDPUKVRQKWBSPK-ZOQUXTDFSA-N 3-methylcytidine Chemical compound O=C1N(C)C(=N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 RDPUKVRQKWBSPK-ZOQUXTDFSA-N 0.000 description 1
- 101800000504 3C-like protease Proteins 0.000 description 1
- ZLOIGESWDJYCTF-UHFFFAOYSA-N 4-Thiouridine Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=S)C=C1 ZLOIGESWDJYCTF-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- YBBDRHCNZBVLGT-FDDDBJFASA-N 4-amino-1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2-oxopyrimidine-5-carbaldehyde Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C(C=O)=C1 YBBDRHCNZBVLGT-FDDDBJFASA-N 0.000 description 1
- OCMSXKMNYAHJMU-JXOAFFINSA-N 4-amino-1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2-oxopyrimidine-5-carbaldehyde Chemical compound C1=C(C=O)C(N)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 OCMSXKMNYAHJMU-JXOAFFINSA-N 0.000 description 1
- WROTXLSEMHAZEA-UHFFFAOYSA-N 4-diaminophosphoryloxymorpholine Chemical compound NP(N)(=O)ON1CCOCC1 WROTXLSEMHAZEA-UHFFFAOYSA-N 0.000 description 1
- CNVRVGAACYEOQI-FDDDBJFASA-N 5,2'-O-dimethylcytidine Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(N)C(C)=C1 CNVRVGAACYEOQI-FDDDBJFASA-N 0.000 description 1
- YHRRPHCORALGKQ-UHFFFAOYSA-N 5,2'-O-dimethyluridine Chemical compound COC1C(O)C(CO)OC1N1C(=O)NC(=O)C(C)=C1 YHRRPHCORALGKQ-UHFFFAOYSA-N 0.000 description 1
- UVGCZRPOXXYZKH-QADQDURISA-N 5-(carboxyhydroxymethyl)uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(C(O)C(O)=O)=C1 UVGCZRPOXXYZKH-QADQDURISA-N 0.000 description 1
- FAWQJBLSWXIJLA-VPCXQMTMSA-N 5-(carboxymethyl)uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(CC(O)=O)=C1 FAWQJBLSWXIJLA-VPCXQMTMSA-N 0.000 description 1
- NFEXJLMYXXIWPI-JXOAFFINSA-N 5-Hydroxymethylcytidine Chemical compound C1=C(CO)C(N)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NFEXJLMYXXIWPI-JXOAFFINSA-N 0.000 description 1
- ZYEWPVTXYBLWRT-UHFFFAOYSA-N 5-Uridinacetamid Natural products O=C1NC(=O)C(CC(=O)N)=CN1C1C(O)C(O)C(CO)O1 ZYEWPVTXYBLWRT-UHFFFAOYSA-N 0.000 description 1
- LOEDKMLIGFMQKR-JXOAFFINSA-N 5-aminomethyl-2-thiouridine Chemical compound S=C1NC(=O)C(CN)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 LOEDKMLIGFMQKR-JXOAFFINSA-N 0.000 description 1
- ZYEWPVTXYBLWRT-VPCXQMTMSA-N 5-carbamoylmethyluridine Chemical compound O=C1NC(=O)C(CC(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZYEWPVTXYBLWRT-VPCXQMTMSA-N 0.000 description 1
- ZXIATBNUWJBBGT-JXOAFFINSA-N 5-methoxyuridine Chemical compound O=C1NC(=O)C(OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZXIATBNUWJBBGT-JXOAFFINSA-N 0.000 description 1
- ZLAQATDNGLKIEV-UHFFFAOYSA-N 5-methyl-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound CC1=CNC(=S)NC1=O ZLAQATDNGLKIEV-UHFFFAOYSA-N 0.000 description 1
- HXVKEKIORVUWDR-UHFFFAOYSA-N 5-methylaminomethyl-2-thiouridine Natural products S=C1NC(=O)C(CNC)=CN1C1C(O)C(O)C(CO)O1 HXVKEKIORVUWDR-UHFFFAOYSA-N 0.000 description 1
- ZXQHKBUIXRFZBV-FDDDBJFASA-N 5-methylaminomethyluridine Chemical compound O=C1NC(=O)C(CNC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 ZXQHKBUIXRFZBV-FDDDBJFASA-N 0.000 description 1
- UJBCLAXPPIDQEE-UHFFFAOYSA-N 5-prop-1-ynyl-1h-pyrimidine-2,4-dione Chemical compound CC#CC1=CNC(=O)NC1=O UJBCLAXPPIDQEE-UHFFFAOYSA-N 0.000 description 1
- KXBCLNRMQPRVTP-UHFFFAOYSA-N 6-amino-1,5-dihydroimidazo[4,5-c]pyridin-4-one Chemical compound O=C1NC(N)=CC2=C1N=CN2 KXBCLNRMQPRVTP-UHFFFAOYSA-N 0.000 description 1
- DCPSTSVLRXOYGS-UHFFFAOYSA-N 6-amino-1h-pyrimidine-2-thione Chemical compound NC1=CC=NC(S)=N1 DCPSTSVLRXOYGS-UHFFFAOYSA-N 0.000 description 1
- QNNARSZPGNJZIX-UHFFFAOYSA-N 6-amino-5-prop-1-ynyl-1h-pyrimidin-2-one Chemical compound CC#CC1=CNC(=O)N=C1N QNNARSZPGNJZIX-UHFFFAOYSA-N 0.000 description 1
- NJBMMMJOXRZENQ-UHFFFAOYSA-N 6H-pyrrolo[2,3-f]quinoline Chemical compound c1cc2ccc3[nH]cccc3c2n1 NJBMMMJOXRZENQ-UHFFFAOYSA-N 0.000 description 1
- MEYMBLGOKYDGLZ-UHFFFAOYSA-N 7-aminomethyl-7-deazaguanine Chemical compound N1=C(N)NC(=O)C2=C1NC=C2CN MEYMBLGOKYDGLZ-UHFFFAOYSA-N 0.000 description 1
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 1
- OGHAROSJZRTIOK-KQYNXXCUSA-O 7-methylguanosine Chemical compound C1=2N=C(N)NC(=O)C=2[N+](C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-O 0.000 description 1
- HBAQYPYDRFILMT-UHFFFAOYSA-N 8-[3-(1-cyclopropylpyrazol-4-yl)-1H-pyrazolo[4,3-d]pyrimidin-5-yl]-3-methyl-3,8-diazabicyclo[3.2.1]octan-2-one Chemical class C1(CC1)N1N=CC(=C1)C1=NNC2=C1N=C(N=C2)N1C2C(N(CC1CC2)C)=O HBAQYPYDRFILMT-UHFFFAOYSA-N 0.000 description 1
- HRYKDUPGBWLLHO-UHFFFAOYSA-N 8-azaadenine Chemical compound NC1=NC=NC2=NNN=C12 HRYKDUPGBWLLHO-UHFFFAOYSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- 229960005508 8-azaguanine Drugs 0.000 description 1
- JSRIPIORIMCGTG-WOUKDFQISA-N 9-[(2R,3R,4R,5R)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-1-methylpurin-6-one Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CN(C)C2=O)=C2N=C1 JSRIPIORIMCGTG-WOUKDFQISA-N 0.000 description 1
- OJTAZBNWKTYVFJ-IOSLPCCCSA-N 9-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2-(methylamino)-3h-purin-6-one Chemical compound C1=2NC(NC)=NC(=O)C=2N=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1OC OJTAZBNWKTYVFJ-IOSLPCCCSA-N 0.000 description 1
- PXBWLHQLSCMJEM-IOSLPCCCSA-N 9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)-2-methyloxolan-2-yl]-3h-purin-6-one Chemical compound C1=NC2=C(O)N=CN=C2N1[C@]1(C)O[C@H](CO)[C@@H](O)[C@H]1O PXBWLHQLSCMJEM-IOSLPCCCSA-N 0.000 description 1
- 108091092742 A-DNA Proteins 0.000 description 1
- 208000035657 Abasia Diseases 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 101710159080 Aconitate hydratase A Proteins 0.000 description 1
- 101710159078 Aconitate hydratase B Proteins 0.000 description 1
- 239000004925 Acrylic resin Substances 0.000 description 1
- 229920000178 Acrylic resin Polymers 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 1
- 102100025976 Adenosine deaminase 2 Human genes 0.000 description 1
- NRCXNPKDOMYPPJ-HYORBCNSSA-N Aflatoxin P1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)O)C=2OC(=O)C2=C1CCC2=O NRCXNPKDOMYPPJ-HYORBCNSSA-N 0.000 description 1
- 208000031277 Amaurotic familial idiocy Diseases 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 101000725156 Aplysia californica Cerebral peptide 1 Proteins 0.000 description 1
- 101000745634 Aplysia californica Cytoplasmic polyadenylation element-binding protein Proteins 0.000 description 1
- 102100021569 Apoptosis regulator Bcl-2 Human genes 0.000 description 1
- 101100165032 Arabidopsis thaliana AZF1 gene Proteins 0.000 description 1
- 101100165034 Arabidopsis thaliana AZF2 gene Proteins 0.000 description 1
- 101100004644 Arabidopsis thaliana BAT1 gene Proteins 0.000 description 1
- 101000719121 Arabidopsis thaliana Protein MEI2-like 1 Proteins 0.000 description 1
- 101000797612 Arabidopsis thaliana Protein MEI2-like 3 Proteins 0.000 description 1
- PEMQXWCOMFJRLS-UHFFFAOYSA-N Archaeosine Natural products C1=2NC(N)=NC(=O)C=2C(C(=N)N)=CN1C1OC(CO)C(O)C1O PEMQXWCOMFJRLS-UHFFFAOYSA-N 0.000 description 1
- 101150010353 Ascl1 gene Proteins 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101000606895 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Pectin lyase 2 Proteins 0.000 description 1
- 101001082391 Aspergillus oryzae Beta-hexosaminidase Proteins 0.000 description 1
- 206010003594 Ataxia telangiectasia Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 108020003591 B-Form DNA Proteins 0.000 description 1
- 102100021631 B-cell lymphoma 6 protein Human genes 0.000 description 1
- 101150104873 BARHL1 gene Proteins 0.000 description 1
- 108700020463 BRCA1 Proteins 0.000 description 1
- 101150072950 BRCA1 gene Proteins 0.000 description 1
- 101150057523 Barhl2 gene Proteins 0.000 description 1
- 101150017888 Bcl2 gene Proteins 0.000 description 1
- 108060000903 Beta-catenin Proteins 0.000 description 1
- 102000015735 Beta-catenin Human genes 0.000 description 1
- 101150040844 Bin1 gene Proteins 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100025401 Breast cancer type 1 susceptibility protein Human genes 0.000 description 1
- 239000002126 C01EB10 - Adenosine Substances 0.000 description 1
- 108010026988 CCAAT-Binding Factor Proteins 0.000 description 1
- 108010014064 CCCTC-Binding Factor Proteins 0.000 description 1
- 102100033849 CCHC-type zinc finger nucleic acid binding protein Human genes 0.000 description 1
- 101710116319 CCHC-type zinc finger nucleic acid binding protein Proteins 0.000 description 1
- 101150035324 CDK9 gene Proteins 0.000 description 1
- 108010083123 CDX2 Transcription Factor Proteins 0.000 description 1
- 101710188750 COUP transcription factor 2 Proteins 0.000 description 1
- 108010018842 CTF-1 transcription factor Proteins 0.000 description 1
- 101100026251 Caenorhabditis elegans atf-2 gene Proteins 0.000 description 1
- 101100170001 Caenorhabditis elegans ddb-1 gene Proteins 0.000 description 1
- 101100227322 Caenorhabditis elegans fli-1 gene Proteins 0.000 description 1
- 101100280477 Caenorhabditis elegans lbp-1 gene Proteins 0.000 description 1
- 101100129500 Caenorhabditis elegans max-2 gene Proteins 0.000 description 1
- 101100518995 Caenorhabditis elegans pax-3 gene Proteins 0.000 description 1
- 101100258233 Caenorhabditis elegans sun-1 gene Proteins 0.000 description 1
- 241000282832 Camelidae Species 0.000 description 1
- 101000841393 Candida albicans Probable NADPH dehydrogenase Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 101100341660 Canis lupus familiaris KRT1 gene Proteins 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 101000850997 Cavia porcellus Eosinophil granule major basic protein 2 Proteins 0.000 description 1
- 101150096994 Cdx1 gene Proteins 0.000 description 1
- 101710082464 Cis-aconitate decarboxylase Proteins 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108010079362 Core Binding Factor Alpha 3 Subunit Proteins 0.000 description 1
- 241000711573 Coronaviridae Species 0.000 description 1
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 1
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 1
- 101710182029 Cyclic AMP-dependent transcription factor ATF-4 Proteins 0.000 description 1
- 101710128030 Cyclic AMP-responsive element-binding protein 5 Proteins 0.000 description 1
- 108010068192 Cyclin A Proteins 0.000 description 1
- 102000002435 Cyclin T Human genes 0.000 description 1
- 102100025191 Cyclin-A2 Human genes 0.000 description 1
- 108010025464 Cyclin-Dependent Kinase 4 Proteins 0.000 description 1
- 102100024112 Cyclin-T2 Human genes 0.000 description 1
- 102100036252 Cyclin-dependent kinase 4 Human genes 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 108091008102 DNA aptamers Proteins 0.000 description 1
- 101710147299 DNA fragmentation factor subunit beta Proteins 0.000 description 1
- 101710096438 DNA-binding protein Proteins 0.000 description 1
- 102100037799 DNA-binding protein Ikaros Human genes 0.000 description 1
- 102100022812 DNA-binding protein RFX2 Human genes 0.000 description 1
- 101100460842 Danio rerio nr2f5 gene Proteins 0.000 description 1
- 101100480530 Danio rerio tal1 gene Proteins 0.000 description 1
- 102100028559 Death domain-associated protein 6 Human genes 0.000 description 1
- 101710085792 Defensin-like protein 1 Proteins 0.000 description 1
- 101001058087 Dictyostelium discoideum Endonuclease 4 homolog Proteins 0.000 description 1
- 101100226017 Dictyostelium discoideum repD gene Proteins 0.000 description 1
- 108010003661 Distal-less homeobox proteins Proteins 0.000 description 1
- 102000004648 Distal-less homeobox proteins Human genes 0.000 description 1
- 102100021212 Double homeobox protein 1 Human genes 0.000 description 1
- 102100021158 Double homeobox protein 4 Human genes 0.000 description 1
- 101000831686 Drosophila melanogaster Protein cycle Proteins 0.000 description 1
- 206010013801 Duchenne Muscular Dystrophy Diseases 0.000 description 1
- 108010036466 E2F2 Transcription Factor Proteins 0.000 description 1
- 108010016085 E2F4 Transcription Factor Proteins 0.000 description 1
- 102100023227 E3 SUMO-protein ligase EGR2 Human genes 0.000 description 1
- 102100034597 E3 ubiquitin-protein ligase TRIM22 Human genes 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 101150060236 EF1 gene Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 101150105460 ERCC2 gene Proteins 0.000 description 1
- 108010032363 ERRalpha estrogen-related receptor Proteins 0.000 description 1
- 102100039578 ETS translocation variant 4 Human genes 0.000 description 1
- 102100023226 Early growth response protein 1 Human genes 0.000 description 1
- 102100021717 Early growth response protein 3 Human genes 0.000 description 1
- 201000011001 Ebola Hemorrhagic Fever Diseases 0.000 description 1
- 208000030820 Ebola disease Diseases 0.000 description 1
- 102100030209 Elongin-B Human genes 0.000 description 1
- 101710191209 Elongin-B Proteins 0.000 description 1
- 102100037114 Elongin-C Human genes 0.000 description 1
- 108050009447 Elongin-C Proteins 0.000 description 1
- 102100032450 Endothelial differentiation-related factor 1 Human genes 0.000 description 1
- 101710182961 Endothelial differentiation-related factor 1 Proteins 0.000 description 1
- 101710100588 Erythroid transcription factor Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101150043847 FOXD1 gene Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010008599 Forkhead Box Protein M1 Proteins 0.000 description 1
- 102100021109 Forkhead box protein B1 Human genes 0.000 description 1
- 102100021084 Forkhead box protein C1 Human genes 0.000 description 1
- 102100021083 Forkhead box protein C2 Human genes 0.000 description 1
- 102100037057 Forkhead box protein D1 Human genes 0.000 description 1
- 102100037062 Forkhead box protein D2 Human genes 0.000 description 1
- 102100037060 Forkhead box protein D3 Human genes 0.000 description 1
- 102100037043 Forkhead box protein D4 Human genes 0.000 description 1
- 102100037042 Forkhead box protein E1 Human genes 0.000 description 1
- 102100020855 Forkhead box protein E3 Human genes 0.000 description 1
- 102100020856 Forkhead box protein F1 Human genes 0.000 description 1
- 102100020848 Forkhead box protein F2 Human genes 0.000 description 1
- 102100041002 Forkhead box protein H1 Human genes 0.000 description 1
- 102100041001 Forkhead box protein I1 Human genes 0.000 description 1
- 102100035128 Forkhead box protein J3 Human genes 0.000 description 1
- 102100035120 Forkhead box protein L1 Human genes 0.000 description 1
- 102100023371 Forkhead box protein N1 Human genes 0.000 description 1
- 102100023360 Forkhead box protein N2 Human genes 0.000 description 1
- 102100023359 Forkhead box protein N3 Human genes 0.000 description 1
- 102100035421 Forkhead box protein O3 Human genes 0.000 description 1
- 102100035416 Forkhead box protein O4 Human genes 0.000 description 1
- 102100028122 Forkhead box protein P1 Human genes 0.000 description 1
- 102100027581 Forkhead box protein P3 Human genes 0.000 description 1
- 102100029346 Forkhead box protein S1 Human genes 0.000 description 1
- 101150096607 Fosl2 gene Proteins 0.000 description 1
- 230000010190 G1 phase Effects 0.000 description 1
- 230000010337 G2 phase Effects 0.000 description 1
- 101710082961 GATA-binding factor 2 Proteins 0.000 description 1
- 102000008412 GATA5 Transcription Factor Human genes 0.000 description 1
- 108010021779 GATA5 Transcription Factor Proteins 0.000 description 1
- 229940126656 GS-4224 Drugs 0.000 description 1
- 101001066288 Gallus gallus GATA-binding factor 3 Proteins 0.000 description 1
- 101000597041 Gallus gallus Transcriptional enhancer factor TEF-3 Proteins 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 102100038073 General transcription factor II-I Human genes 0.000 description 1
- 101710144827 General transcription factor II-I Proteins 0.000 description 1
- 102100034936 General transcription factor IIE subunit 1 Human genes 0.000 description 1
- 101710202045 General transcription factor IIF subunit 1 Proteins 0.000 description 1
- 102100033842 General transcription factor IIF subunit 2 Human genes 0.000 description 1
- 101710202044 General transcription factor IIF subunit 2 Proteins 0.000 description 1
- 108091010837 Glial cell line-derived neurotrophic factor Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010053759 Growth retardation Diseases 0.000 description 1
- 101150032426 HLF gene Proteins 0.000 description 1
- 102000049982 HMGA2 Human genes 0.000 description 1
- 108700039143 HMGA2 Proteins 0.000 description 1
- 102100034049 Heat shock factor protein 2 Human genes 0.000 description 1
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 1
- 102100031880 Helicase SRCAP Human genes 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 108010061414 Hepatocyte Nuclear Factor 1-beta Proteins 0.000 description 1
- 108010038661 Hepatocyte Nuclear Factor 3-alpha Proteins 0.000 description 1
- 102000010818 Hepatocyte Nuclear Factor 3-alpha Human genes 0.000 description 1
- 108010087745 Hepatocyte Nuclear Factor 3-beta Proteins 0.000 description 1
- 102000009094 Hepatocyte Nuclear Factor 3-beta Human genes 0.000 description 1
- 108010055480 Hepatocyte Nuclear Factor 3-gamma Proteins 0.000 description 1
- 102000000155 Hepatocyte Nuclear Factor 3-gamma Human genes 0.000 description 1
- 102100022123 Hepatocyte nuclear factor 1-beta Human genes 0.000 description 1
- 102100022054 Hepatocyte nuclear factor 4-alpha Human genes 0.000 description 1
- 102000005646 Heterogeneous-Nuclear Ribonucleoprotein K Human genes 0.000 description 1
- 108010084680 Heterogeneous-Nuclear Ribonucleoprotein K Proteins 0.000 description 1
- 101710189113 Histone H4 transcription factor Proteins 0.000 description 1
- 102100030445 Histone H4 transcription factor Human genes 0.000 description 1
- 102100039996 Histone deacetylase 1 Human genes 0.000 description 1
- 102100039999 Histone deacetylase 2 Human genes 0.000 description 1
- 102100021455 Histone deacetylase 3 Human genes 0.000 description 1
- 102100022103 Histone-lysine N-methyltransferase 2A Human genes 0.000 description 1
- 108050002855 Histone-lysine N-methyltransferase 2A Proteins 0.000 description 1
- 102100038970 Histone-lysine N-methyltransferase EZH2 Human genes 0.000 description 1
- 102100032742 Histone-lysine N-methyltransferase SETD2 Human genes 0.000 description 1
- 101150022826 Hnf4g gene Proteins 0.000 description 1
- 102100031671 Homeobox protein CDX-2 Human genes 0.000 description 1
- 102100030309 Homeobox protein Hox-A1 Human genes 0.000 description 1
- 102100030308 Homeobox protein Hox-A11 Human genes 0.000 description 1
- 102100030307 Homeobox protein Hox-A13 Human genes 0.000 description 1
- 102100039542 Homeobox protein Hox-A2 Human genes 0.000 description 1
- 102100039541 Homeobox protein Hox-A3 Human genes 0.000 description 1
- 102100025116 Homeobox protein Hox-A4 Human genes 0.000 description 1
- 102100022649 Homeobox protein Hox-A6 Human genes 0.000 description 1
- 102100022650 Homeobox protein Hox-A7 Human genes 0.000 description 1
- 102100021088 Homeobox protein Hox-B13 Human genes 0.000 description 1
- 102100034862 Homeobox protein Hox-B2 Human genes 0.000 description 1
- 102100028411 Homeobox protein Hox-B3 Human genes 0.000 description 1
- 102100028404 Homeobox protein Hox-B4 Human genes 0.000 description 1
- 102100029240 Homeobox protein Hox-B5 Human genes 0.000 description 1
- 102100025056 Homeobox protein Hox-B6 Human genes 0.000 description 1
- 102100025061 Homeobox protein Hox-B7 Human genes 0.000 description 1
- 102100029423 Homeobox protein Hox-B8 Human genes 0.000 description 1
- 102100029433 Homeobox protein Hox-B9 Human genes 0.000 description 1
- 102100029426 Homeobox protein Hox-C10 Human genes 0.000 description 1
- 102100020766 Homeobox protein Hox-C11 Human genes 0.000 description 1
- 102100020758 Homeobox protein Hox-C12 Human genes 0.000 description 1
- 102100020761 Homeobox protein Hox-C13 Human genes 0.000 description 1
- 102100020759 Homeobox protein Hox-C4 Human genes 0.000 description 1
- 102100020762 Homeobox protein Hox-C5 Human genes 0.000 description 1
- 102100022599 Homeobox protein Hox-C6 Human genes 0.000 description 1
- 102100022601 Homeobox protein Hox-C8 Human genes 0.000 description 1
- 102100022597 Homeobox protein Hox-C9 Human genes 0.000 description 1
- 102100039544 Homeobox protein Hox-D10 Human genes 0.000 description 1
- 102100039545 Homeobox protein Hox-D11 Human genes 0.000 description 1
- 102100040205 Homeobox protein Hox-D12 Human genes 0.000 description 1
- 102100040227 Homeobox protein Hox-D13 Human genes 0.000 description 1
- 102100040228 Homeobox protein Hox-D3 Human genes 0.000 description 1
- 102100021086 Homeobox protein Hox-D4 Human genes 0.000 description 1
- 102100034858 Homeobox protein Hox-D8 Human genes 0.000 description 1
- 102100034864 Homeobox protein Hox-D9 Human genes 0.000 description 1
- 102100027893 Homeobox protein Nkx-2.1 Human genes 0.000 description 1
- 101710114425 Homeobox protein Nkx-2.1 Proteins 0.000 description 1
- 102100027886 Homeobox protein Nkx-2.2 Human genes 0.000 description 1
- 102100027890 Homeobox protein Nkx-2.3 Human genes 0.000 description 1
- 102100027875 Homeobox protein Nkx-2.5 Human genes 0.000 description 1
- 102100027877 Homeobox protein Nkx-2.8 Human genes 0.000 description 1
- 102100028091 Homeobox protein Nkx-3.2 Human genes 0.000 description 1
- 102100028098 Homeobox protein Nkx-6.1 Human genes 0.000 description 1
- 102100029394 Homeobox protein PKNOX1 Human genes 0.000 description 1
- 102100035081 Homeobox protein TGIF1 Human genes 0.000 description 1
- 102100035082 Homeobox protein TGIF2 Human genes 0.000 description 1
- 102100039704 Homeobox protein VENTX Human genes 0.000 description 1
- 102100030234 Homeobox protein cut-like 1 Human genes 0.000 description 1
- 101000866618 Homo sapiens 3-beta-hydroxysteroid-Delta(8),Delta(7)-isomerase Proteins 0.000 description 1
- 101000718065 Homo sapiens AKT-interacting protein Proteins 0.000 description 1
- 101000720051 Homo sapiens Adenosine deaminase 2 Proteins 0.000 description 1
- 101000800875 Homo sapiens Alpha-globin transcription factor CP2 Proteins 0.000 description 1
- 101000971234 Homo sapiens B-cell lymphoma 6 protein Proteins 0.000 description 1
- 101000765010 Homo sapiens Beta-galactosidase Proteins 0.000 description 1
- 101000860860 Homo sapiens COUP transcription factor 2 Proteins 0.000 description 1
- 101000599038 Homo sapiens DNA-binding protein Ikaros Proteins 0.000 description 1
- 101000756799 Homo sapiens DNA-binding protein RFX2 Proteins 0.000 description 1
- 101000915428 Homo sapiens Death domain-associated protein 6 Proteins 0.000 description 1
- 101000968544 Homo sapiens Double homeobox protein 1 Proteins 0.000 description 1
- 101000968549 Homo sapiens Double homeobox protein 4 Proteins 0.000 description 1
- 101001049692 Homo sapiens E3 SUMO-protein ligase EGR2 Proteins 0.000 description 1
- 101000636713 Homo sapiens E3 ubiquitin-protein ligase NEDD4 Proteins 0.000 description 1
- 101000848629 Homo sapiens E3 ubiquitin-protein ligase TRIM22 Proteins 0.000 description 1
- 101000813747 Homo sapiens ETS translocation variant 4 Proteins 0.000 description 1
- 101001049697 Homo sapiens Early growth response protein 1 Proteins 0.000 description 1
- 101000896450 Homo sapiens Early growth response protein 3 Proteins 0.000 description 1
- 101001066268 Homo sapiens Erythroid transcription factor Proteins 0.000 description 1
- 101000818727 Homo sapiens Forkhead box protein B1 Proteins 0.000 description 1
- 101000818310 Homo sapiens Forkhead box protein C1 Proteins 0.000 description 1
- 101000818305 Homo sapiens Forkhead box protein C2 Proteins 0.000 description 1
- 101001029314 Homo sapiens Forkhead box protein D2 Proteins 0.000 description 1
- 101001029308 Homo sapiens Forkhead box protein D3 Proteins 0.000 description 1
- 101001029302 Homo sapiens Forkhead box protein D4 Proteins 0.000 description 1
- 101001029304 Homo sapiens Forkhead box protein E1 Proteins 0.000 description 1
- 101000931489 Homo sapiens Forkhead box protein E3 Proteins 0.000 description 1
- 101000931494 Homo sapiens Forkhead box protein F1 Proteins 0.000 description 1
- 101000931482 Homo sapiens Forkhead box protein F2 Proteins 0.000 description 1
- 101000892840 Homo sapiens Forkhead box protein H1 Proteins 0.000 description 1
- 101000892875 Homo sapiens Forkhead box protein I1 Proteins 0.000 description 1
- 101001023387 Homo sapiens Forkhead box protein J3 Proteins 0.000 description 1
- 101001023352 Homo sapiens Forkhead box protein L1 Proteins 0.000 description 1
- 101000907576 Homo sapiens Forkhead box protein N1 Proteins 0.000 description 1
- 101000907593 Homo sapiens Forkhead box protein N2 Proteins 0.000 description 1
- 101000907594 Homo sapiens Forkhead box protein N3 Proteins 0.000 description 1
- 101000877681 Homo sapiens Forkhead box protein O3 Proteins 0.000 description 1
- 101000877683 Homo sapiens Forkhead box protein O4 Proteins 0.000 description 1
- 101001059893 Homo sapiens Forkhead box protein P1 Proteins 0.000 description 1
- 101000861452 Homo sapiens Forkhead box protein P3 Proteins 0.000 description 1
- 101001062403 Homo sapiens Forkhead box protein S1 Proteins 0.000 description 1
- 101000876511 Homo sapiens General transcription and DNA repair factor IIH helicase subunit XPD Proteins 0.000 description 1
- 101001016883 Homo sapiens Heat shock factor protein 2 Proteins 0.000 description 1
- 101001016865 Homo sapiens Heat shock protein HSP 90-alpha Proteins 0.000 description 1
- 101000704158 Homo sapiens Helicase SRCAP Proteins 0.000 description 1
- 101001045740 Homo sapiens Hepatocyte nuclear factor 4-alpha Proteins 0.000 description 1
- 101001035024 Homo sapiens Histone deacetylase 1 Proteins 0.000 description 1
- 101001035011 Homo sapiens Histone deacetylase 2 Proteins 0.000 description 1
- 101000899282 Homo sapiens Histone deacetylase 3 Proteins 0.000 description 1
- 101000882127 Homo sapiens Histone-lysine N-methyltransferase EZH2 Proteins 0.000 description 1
- 101000654725 Homo sapiens Histone-lysine N-methyltransferase SETD2 Proteins 0.000 description 1
- 101000901614 Homo sapiens Homeobox protein DLX-4 Proteins 0.000 description 1
- 101001083156 Homo sapiens Homeobox protein Hox-A1 Proteins 0.000 description 1
- 101001083158 Homo sapiens Homeobox protein Hox-A11 Proteins 0.000 description 1
- 101000962636 Homo sapiens Homeobox protein Hox-A2 Proteins 0.000 description 1
- 101000962622 Homo sapiens Homeobox protein Hox-A3 Proteins 0.000 description 1
- 101001077578 Homo sapiens Homeobox protein Hox-A4 Proteins 0.000 description 1
- 101001045083 Homo sapiens Homeobox protein Hox-A6 Proteins 0.000 description 1
- 101001045116 Homo sapiens Homeobox protein Hox-A7 Proteins 0.000 description 1
- 101001041145 Homo sapiens Homeobox protein Hox-B13 Proteins 0.000 description 1
- 101001019752 Homo sapiens Homeobox protein Hox-B2 Proteins 0.000 description 1
- 101000839775 Homo sapiens Homeobox protein Hox-B3 Proteins 0.000 description 1
- 101000839788 Homo sapiens Homeobox protein Hox-B4 Proteins 0.000 description 1
- 101000840553 Homo sapiens Homeobox protein Hox-B5 Proteins 0.000 description 1
- 101001077542 Homo sapiens Homeobox protein Hox-B6 Proteins 0.000 description 1
- 101001077539 Homo sapiens Homeobox protein Hox-B7 Proteins 0.000 description 1
- 101000988994 Homo sapiens Homeobox protein Hox-B8 Proteins 0.000 description 1
- 101000989000 Homo sapiens Homeobox protein Hox-B9 Proteins 0.000 description 1
- 101000989027 Homo sapiens Homeobox protein Hox-C10 Proteins 0.000 description 1
- 101001003015 Homo sapiens Homeobox protein Hox-C11 Proteins 0.000 description 1
- 101001002991 Homo sapiens Homeobox protein Hox-C12 Proteins 0.000 description 1
- 101001002988 Homo sapiens Homeobox protein Hox-C13 Proteins 0.000 description 1
- 101001002994 Homo sapiens Homeobox protein Hox-C4 Proteins 0.000 description 1
- 101001002966 Homo sapiens Homeobox protein Hox-C5 Proteins 0.000 description 1
- 101001045154 Homo sapiens Homeobox protein Hox-C6 Proteins 0.000 description 1
- 101001045158 Homo sapiens Homeobox protein Hox-C8 Proteins 0.000 description 1
- 101001045140 Homo sapiens Homeobox protein Hox-C9 Proteins 0.000 description 1
- 101000962573 Homo sapiens Homeobox protein Hox-D10 Proteins 0.000 description 1
- 101000962591 Homo sapiens Homeobox protein Hox-D11 Proteins 0.000 description 1
- 101001037169 Homo sapiens Homeobox protein Hox-D12 Proteins 0.000 description 1
- 101001037168 Homo sapiens Homeobox protein Hox-D13 Proteins 0.000 description 1
- 101001037158 Homo sapiens Homeobox protein Hox-D3 Proteins 0.000 description 1
- 101001041136 Homo sapiens Homeobox protein Hox-D4 Proteins 0.000 description 1
- 101001019776 Homo sapiens Homeobox protein Hox-D8 Proteins 0.000 description 1
- 101001019766 Homo sapiens Homeobox protein Hox-D9 Proteins 0.000 description 1
- 101000632186 Homo sapiens Homeobox protein Nkx-2.2 Proteins 0.000 description 1
- 101000632181 Homo sapiens Homeobox protein Nkx-2.3 Proteins 0.000 description 1
- 101000632197 Homo sapiens Homeobox protein Nkx-2.5 Proteins 0.000 description 1
- 101000578251 Homo sapiens Homeobox protein Nkx-3.2 Proteins 0.000 description 1
- 101000578254 Homo sapiens Homeobox protein Nkx-6.1 Proteins 0.000 description 1
- 101001125957 Homo sapiens Homeobox protein PKNOX1 Proteins 0.000 description 1
- 101000596925 Homo sapiens Homeobox protein TGIF1 Proteins 0.000 description 1
- 101000596938 Homo sapiens Homeobox protein TGIF2 Proteins 0.000 description 1
- 101000667986 Homo sapiens Homeobox protein VENTX Proteins 0.000 description 1
- 101000726740 Homo sapiens Homeobox protein cut-like 1 Proteins 0.000 description 1
- 101001083543 Homo sapiens Host cell factor 1 Proteins 0.000 description 1
- 101001021527 Homo sapiens Huntingtin-interacting protein 1 Proteins 0.000 description 1
- 101001001462 Homo sapiens Importin subunit alpha-5 Proteins 0.000 description 1
- 101000852539 Homo sapiens Importin-5 Proteins 0.000 description 1
- 101001056180 Homo sapiens Induced myeloid leukemia cell differentiation protein Mcl-1 Proteins 0.000 description 1
- 101001033233 Homo sapiens Interleukin-10 Proteins 0.000 description 1
- 101001139130 Homo sapiens Krueppel-like factor 5 Proteins 0.000 description 1
- 101001090713 Homo sapiens L-lactate dehydrogenase A chain Proteins 0.000 description 1
- 101001022957 Homo sapiens LIM domain-binding protein 1 Proteins 0.000 description 1
- 101001022948 Homo sapiens LIM domain-binding protein 2 Proteins 0.000 description 1
- 101001038339 Homo sapiens LIM homeobox transcription factor 1-alpha Proteins 0.000 description 1
- 101000984044 Homo sapiens LIM homeobox transcription factor 1-beta Proteins 0.000 description 1
- 101001020548 Homo sapiens LIM/homeobox protein Lhx1 Proteins 0.000 description 1
- 101001020544 Homo sapiens LIM/homeobox protein Lhx2 Proteins 0.000 description 1
- 101000624625 Homo sapiens M-phase inducer phosphatase 1 Proteins 0.000 description 1
- 101000576323 Homo sapiens Motor neuron and pancreas homeobox protein 1 Proteins 0.000 description 1
- 101001128495 Homo sapiens Myeloid zinc finger 1 Proteins 0.000 description 1
- 101100460510 Homo sapiens NKX2-8 gene Proteins 0.000 description 1
- 101000979909 Homo sapiens NMDA receptor synaptonuclear signaling and neuronal migration factor Proteins 0.000 description 1
- 101000588302 Homo sapiens Nuclear factor erythroid 2-related factor 2 Proteins 0.000 description 1
- 101000602930 Homo sapiens Nuclear receptor coactivator 2 Proteins 0.000 description 1
- 101000603323 Homo sapiens Nuclear receptor subfamily 0 group B member 1 Proteins 0.000 description 1
- 101000978926 Homo sapiens Nuclear receptor subfamily 1 group D member 1 Proteins 0.000 description 1
- 101000603882 Homo sapiens Nuclear receptor subfamily 1 group I member 3 Proteins 0.000 description 1
- 101000633516 Homo sapiens Nuclear receptor subfamily 2 group F member 6 Proteins 0.000 description 1
- 101001109698 Homo sapiens Nuclear receptor subfamily 4 group A member 2 Proteins 0.000 description 1
- 101001109685 Homo sapiens Nuclear receptor subfamily 5 group A member 2 Proteins 0.000 description 1
- 101000734351 Homo sapiens PDZ and LIM domain protein 1 Proteins 0.000 description 1
- 101000612089 Homo sapiens Pancreas/duodenum homeobox protein 1 Proteins 0.000 description 1
- 101000633511 Homo sapiens Photoreceptor-specific nuclear receptor Proteins 0.000 description 1
- 101000583156 Homo sapiens Pituitary homeobox 1 Proteins 0.000 description 1
- 101000595669 Homo sapiens Pituitary homeobox 2 Proteins 0.000 description 1
- 101000595674 Homo sapiens Pituitary homeobox 3 Proteins 0.000 description 1
- 101000584499 Homo sapiens Polycomb protein SUZ12 Proteins 0.000 description 1
- 101000693750 Homo sapiens Prefoldin subunit 5 Proteins 0.000 description 1
- 101000761460 Homo sapiens Protein CASP Proteins 0.000 description 1
- 101000721172 Homo sapiens Protein DBF4 homolog A Proteins 0.000 description 1
- 101000640050 Homo sapiens Protein strawberry notch homolog 1 Proteins 0.000 description 1
- 101000738322 Homo sapiens Prothymosin alpha Proteins 0.000 description 1
- 101000968552 Homo sapiens Putative double homeobox protein 3 Proteins 0.000 description 1
- 101001093899 Homo sapiens Retinoic acid receptor RXR-alpha Proteins 0.000 description 1
- 101000640876 Homo sapiens Retinoic acid receptor RXR-beta Proteins 0.000 description 1
- 101000703463 Homo sapiens Rho GTPase-activating protein 35 Proteins 0.000 description 1
- 101000754919 Homo sapiens Ribosomal oxygenase 2 Proteins 0.000 description 1
- 101000650547 Homo sapiens Ribosome production factor 1 Proteins 0.000 description 1
- 101000857677 Homo sapiens Runt-related transcription factor 1 Proteins 0.000 description 1
- 101000857682 Homo sapiens Runt-related transcription factor 2 Proteins 0.000 description 1
- 101000694550 Homo sapiens RuvB-like 1 Proteins 0.000 description 1
- 101000616556 Homo sapiens SH3 domain-containing protein 19 Proteins 0.000 description 1
- 101000826130 Homo sapiens Sex-determining region Y protein Proteins 0.000 description 1
- 101000585484 Homo sapiens Signal transducer and activator of transcription 1-alpha/beta Proteins 0.000 description 1
- 101000897669 Homo sapiens Small RNA 2'-O-methyltransferase Proteins 0.000 description 1
- 101000868152 Homo sapiens Son of sevenless homolog 1 Proteins 0.000 description 1
- 101000851696 Homo sapiens Steroid hormone receptor ERR2 Proteins 0.000 description 1
- 101000625913 Homo sapiens T-box transcription factor TBX4 Proteins 0.000 description 1
- 101000800488 Homo sapiens T-cell leukemia homeobox protein 1 Proteins 0.000 description 1
- 101000655118 Homo sapiens T-cell leukemia homeobox protein 2 Proteins 0.000 description 1
- 101000655119 Homo sapiens T-cell leukemia homeobox protein 3 Proteins 0.000 description 1
- 101000694973 Homo sapiens TATA-binding protein-associated factor 172 Proteins 0.000 description 1
- 101000837626 Homo sapiens Thyroid hormone receptor alpha Proteins 0.000 description 1
- 101000663444 Homo sapiens Transcription elongation factor SPT4 Proteins 0.000 description 1
- 101000702364 Homo sapiens Transcription elongation factor SPT5 Proteins 0.000 description 1
- 101000904152 Homo sapiens Transcription factor E2F1 Proteins 0.000 description 1
- 101000946167 Homo sapiens Transcription factor LBX1 Proteins 0.000 description 1
- 101000756787 Homo sapiens Transcription factor RFX3 Proteins 0.000 description 1
- 101000596093 Homo sapiens Transcription initiation factor TFIID subunit 1 Proteins 0.000 description 1
- 101000652707 Homo sapiens Transcription initiation factor TFIID subunit 4 Proteins 0.000 description 1
- 101001074042 Homo sapiens Transcriptional activator GLI3 Proteins 0.000 description 1
- 101000657352 Homo sapiens Transcriptional adapter 2-alpha Proteins 0.000 description 1
- 101000653735 Homo sapiens Transcriptional enhancer factor TEF-1 Proteins 0.000 description 1
- 101000940144 Homo sapiens Transcriptional repressor protein YY1 Proteins 0.000 description 1
- 101000669432 Homo sapiens Transducin-like enhancer protein 1 Proteins 0.000 description 1
- 101000971144 Homo sapiens Tyrosine-protein kinase BAZ1B Proteins 0.000 description 1
- 101000747867 Homo sapiens Upstream-binding protein 1 Proteins 0.000 description 1
- 101000767597 Homo sapiens Vascular endothelial zinc finger 1 Proteins 0.000 description 1
- 101000791652 Homo sapiens YY1-associated factor 2 Proteins 0.000 description 1
- 101100377226 Homo sapiens ZBTB16 gene Proteins 0.000 description 1
- 101000759185 Homo sapiens Zinc finger X-chromosomal protein Proteins 0.000 description 1
- 101000964478 Homo sapiens Zinc finger and BTB domain-containing protein 17 Proteins 0.000 description 1
- 101000818563 Homo sapiens Zinc finger and BTB domain-containing protein 25 Proteins 0.000 description 1
- 101000788840 Homo sapiens Zinc finger and BTB domain-containing protein 6 Proteins 0.000 description 1
- 101000785559 Homo sapiens Zinc finger and SCAN domain-containing protein 26 Proteins 0.000 description 1
- 101000964587 Homo sapiens Zinc finger protein 174 Proteins 0.000 description 1
- 101000976643 Homo sapiens Zinc finger protein ZIC 2 Proteins 0.000 description 1
- 101000788690 Homo sapiens Zinc fingers and homeoboxes protein 1 Proteins 0.000 description 1
- 101000687642 Homo sapiens snRNA-activating protein complex subunit 1 Proteins 0.000 description 1
- 101000687648 Homo sapiens snRNA-activating protein complex subunit 2 Proteins 0.000 description 1
- 101000825856 Homo sapiens snRNA-activating protein complex subunit 3 Proteins 0.000 description 1
- 101000825848 Homo sapiens snRNA-activating protein complex subunit 4 Proteins 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 102100035957 Huntingtin-interacting protein 1 Human genes 0.000 description 1
- 208000023105 Huntington disease Diseases 0.000 description 1
- 208000000563 Hyperlipoproteinemia Type II Diseases 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 102100035692 Importin subunit alpha-1 Human genes 0.000 description 1
- 102100036340 Importin-5 Human genes 0.000 description 1
- 102100026539 Induced myeloid leukemia cell differentiation protein Mcl-1 Human genes 0.000 description 1
- 102100027636 Insulin-like growth factor-binding protein 1 Human genes 0.000 description 1
- 108090000957 Insulin-like growth factor-binding protein 1 Proteins 0.000 description 1
- 108090000890 Interferon regulatory factor 1 Proteins 0.000 description 1
- 102000004289 Interferon regulatory factor 1 Human genes 0.000 description 1
- 102100029838 Interferon regulatory factor 2 Human genes 0.000 description 1
- 108090000908 Interferon regulatory factor 2 Proteins 0.000 description 1
- 102100038069 Interferon regulatory factor 8 Human genes 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108010041872 Islet Amyloid Polypeptide Proteins 0.000 description 1
- 102100027670 Islet amyloid polypeptide Human genes 0.000 description 1
- 101150026829 JUNB gene Proteins 0.000 description 1
- 101150021395 JUND gene Proteins 0.000 description 1
- 108091036429 KCNQ1OT1 Proteins 0.000 description 1
- 101150023743 KLF9 gene Proteins 0.000 description 1
- 102100020678 Krueppel-like factor 3 Human genes 0.000 description 1
- 101710116712 Krueppel-like factor 3 Proteins 0.000 description 1
- 102100020680 Krueppel-like factor 5 Human genes 0.000 description 1
- 102100020679 Krueppel-like factor 6 Human genes 0.000 description 1
- 102100020684 Krueppel-like factor 9 Human genes 0.000 description 1
- 108010049058 Kruppel-Like Factor 6 Proteins 0.000 description 1
- 102000015335 Ku Autoantigen Human genes 0.000 description 1
- 108010025026 Ku Autoantigen Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 102100034671 L-lactate dehydrogenase A chain Human genes 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 102100035114 LIM domain-binding protein 1 Human genes 0.000 description 1
- 102100040290 LIM homeobox transcription factor 1-alpha Human genes 0.000 description 1
- 102100025457 LIM homeobox transcription factor 1-beta Human genes 0.000 description 1
- 102100036133 LIM/homeobox protein Lhx1 Human genes 0.000 description 1
- 102100036132 LIM/homeobox protein Lhx2 Human genes 0.000 description 1
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 1
- 102100022699 Lymphoid enhancer-binding factor 1 Human genes 0.000 description 1
- 108090001093 Lymphoid enhancer-binding factor 1 Proteins 0.000 description 1
- 102100023326 M-phase inducer phosphatase 1 Human genes 0.000 description 1
- 108010064699 MSH Release-Inhibiting Hormone Proteins 0.000 description 1
- 101150078498 MYB gene Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108091027974 Mature messenger RNA Proteins 0.000 description 1
- NOOJLZTTWSNHOX-UWVGGRQHSA-N Melanostatin Chemical compound NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 NOOJLZTTWSNHOX-UWVGGRQHSA-N 0.000 description 1
- 108090000192 Methionyl aminopeptidases Proteins 0.000 description 1
- 108700011259 MicroRNAs Proteins 0.000 description 1
- 102100025744 Mothers against decapentaplegic homolog 1 Human genes 0.000 description 1
- 102100025751 Mothers against decapentaplegic homolog 2 Human genes 0.000 description 1
- 101710143123 Mothers against decapentaplegic homolog 2 Proteins 0.000 description 1
- 102100025748 Mothers against decapentaplegic homolog 3 Human genes 0.000 description 1
- 101710143111 Mothers against decapentaplegic homolog 3 Proteins 0.000 description 1
- 102100025725 Mothers against decapentaplegic homolog 4 Human genes 0.000 description 1
- 101710143112 Mothers against decapentaplegic homolog 4 Proteins 0.000 description 1
- 102100030610 Mothers against decapentaplegic homolog 5 Human genes 0.000 description 1
- 101710143113 Mothers against decapentaplegic homolog 5 Proteins 0.000 description 1
- 102100025170 Motor neuron and pancreas homeobox protein 1 Human genes 0.000 description 1
- 101150118570 Msx2 gene Proteins 0.000 description 1
- 101100220214 Mus musculus Cdx4 gene Proteins 0.000 description 1
- 101100445099 Mus musculus Emx1 gene Proteins 0.000 description 1
- 101100285407 Mus musculus En1 gene Proteins 0.000 description 1
- 101100285414 Mus musculus En2 gene Proteins 0.000 description 1
- 101100281205 Mus musculus Fli1 gene Proteins 0.000 description 1
- 101100013973 Mus musculus Gata4 gene Proteins 0.000 description 1
- 101100121434 Mus musculus Gcm1 gene Proteins 0.000 description 1
- 101100071843 Mus musculus Hoxb1 gene Proteins 0.000 description 1
- 101100184520 Mus musculus Mnt gene Proteins 0.000 description 1
- 101100024583 Mus musculus Mtf1 gene Proteins 0.000 description 1
- 101100518987 Mus musculus Pax1 gene Proteins 0.000 description 1
- 101100518992 Mus musculus Pax2 gene Proteins 0.000 description 1
- 101100518997 Mus musculus Pax3 gene Proteins 0.000 description 1
- 101100351017 Mus musculus Pax4 gene Proteins 0.000 description 1
- 101100351020 Mus musculus Pax5 gene Proteins 0.000 description 1
- 101100351033 Mus musculus Pax7 gene Proteins 0.000 description 1
- 101100462885 Mus musculus Pax9 gene Proteins 0.000 description 1
- 101100521345 Mus musculus Prop1 gene Proteins 0.000 description 1
- 101100366227 Mus musculus Sox11 gene Proteins 0.000 description 1
- 101100366231 Mus musculus Sox12 gene Proteins 0.000 description 1
- 101100043050 Mus musculus Sox4 gene Proteins 0.000 description 1
- 101100096242 Mus musculus Sox9 gene Proteins 0.000 description 1
- 101100480538 Mus musculus Tal1 gene Proteins 0.000 description 1
- 102100034711 Myb-related protein A Human genes 0.000 description 1
- 101710115158 Myb-related protein A Proteins 0.000 description 1
- 101710115153 Myb-related protein B Proteins 0.000 description 1
- 102100034670 Myb-related protein B Human genes 0.000 description 1
- 108010000591 Myc associated factor X Proteins 0.000 description 1
- 102100031790 Myelin expression factor 2 Human genes 0.000 description 1
- 101710107751 Myelin expression factor 2 Proteins 0.000 description 1
- 108700041619 Myeloid Ecotropic Viral Integration Site 1 Proteins 0.000 description 1
- 102000047831 Myeloid Ecotropic Viral Integration Site 1 Human genes 0.000 description 1
- 102100031827 Myeloid zinc finger 1 Human genes 0.000 description 1
- 101710099061 Myogenic factor 5 Proteins 0.000 description 1
- IYYIBFCJILKPCO-WOUKDFQISA-O N(2),N(2),N(7)-trimethylguanosine Chemical compound C1=2NC(N(C)C)=NC(=O)C=2N(C)C=[N+]1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O IYYIBFCJILKPCO-WOUKDFQISA-O 0.000 description 1
- RSPURTUNRHNVGF-IOSLPCCCSA-N N(2),N(2)-dimethylguanosine Chemical compound C1=NC=2C(=O)NC(N(C)C)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O RSPURTUNRHNVGF-IOSLPCCCSA-N 0.000 description 1
- ZBYRSRLCXTUFLJ-IOSLPCCCSA-O N(2),N(7)-dimethylguanosine Chemical compound CNC=1NC(C=2[N+](=CN([C@H]3[C@H](O)[C@H](O)[C@@H](CO)O3)C=2N=1)C)=O ZBYRSRLCXTUFLJ-IOSLPCCCSA-O 0.000 description 1
- SLEHROROQDYRAW-KQYNXXCUSA-N N(2)-methylguanosine Chemical compound C1=NC=2C(=O)NC(NC)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O SLEHROROQDYRAW-KQYNXXCUSA-N 0.000 description 1
- NIDVTARKFBZMOT-PEBGCTIMSA-N N(4)-acetylcytidine Chemical compound O=C1N=C(NC(=O)C)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NIDVTARKFBZMOT-PEBGCTIMSA-N 0.000 description 1
- WVGPGNPCZPYCLK-WOUKDFQISA-N N(6),N(6)-dimethyladenosine Chemical compound C1=NC=2C(N(C)C)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O WVGPGNPCZPYCLK-WOUKDFQISA-N 0.000 description 1
- USVMJSALORZVDV-SDBHATRESA-N N(6)-(Delta(2)-isopentenyl)adenosine Chemical compound C1=NC=2C(NCC=C(C)C)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O USVMJSALORZVDV-SDBHATRESA-N 0.000 description 1
- OVRNDRQMDRJTHS-CBQIKETKSA-N N-Acetyl-D-Galactosamine Chemical compound CC(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-CBQIKETKSA-N 0.000 description 1
- UNUYMBPXEFMLNW-DWVDDHQFSA-N N-[(9-beta-D-ribofuranosylpurin-6-yl)carbamoyl]threonine Chemical compound C1=NC=2C(NC(=O)N[C@@H]([C@H](O)C)C(O)=O)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O UNUYMBPXEFMLNW-DWVDDHQFSA-N 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- SLLVJTURCPWLTP-UHFFFAOYSA-N N-[9-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]purin-6-yl]acetamide Chemical compound C1=NC=2C(NC(=O)C)=NC=NC=2N1C1OC(CO)C(O)C1O SLLVJTURCPWLTP-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-KEWYIRBNSA-N N-acetyl-D-galactosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@H](O)[C@@H]1O OVRNDRQMDRJTHS-KEWYIRBNSA-N 0.000 description 1
- 102100034449 N-myc-interactor Human genes 0.000 description 1
- 101710190516 N-myc-interactor Proteins 0.000 description 1
- 108010049175 N-substituted Glycines Proteins 0.000 description 1
- LZCNWAXLJWBRJE-ZOQUXTDFSA-N N4-Methylcytidine Chemical compound O=C1N=C(NC)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 LZCNWAXLJWBRJE-ZOQUXTDFSA-N 0.000 description 1
- GOSWTRUMMSCNCW-UHFFFAOYSA-N N6-(cis-hydroxyisopentenyl)adenosine Chemical compound C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1OC(CO)C(O)C1O GOSWTRUMMSCNCW-UHFFFAOYSA-N 0.000 description 1
- 102000018745 NF-KappaB Inhibitor alpha Human genes 0.000 description 1
- 108010052419 NF-KappaB Inhibitor alpha Proteins 0.000 description 1
- 102100024546 NMDA receptor synaptonuclear signaling and neuronal migration factor Human genes 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 101150006407 NRF1 gene Proteins 0.000 description 1
- 208000002454 Nasopharyngeal Carcinoma Diseases 0.000 description 1
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 208000002537 Neuronal Ceroid-Lipofuscinoses Diseases 0.000 description 1
- 101100445499 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) erg-1 gene Proteins 0.000 description 1
- 101100133350 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) nhp-1 gene Proteins 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 101800000353 Non-collagenous domain 1 Proteins 0.000 description 1
- 101150095442 Nr1h2 gene Proteins 0.000 description 1
- 101800000398 Nsp2 cysteine proteinase Proteins 0.000 description 1
- 101710205482 Nuclear factor 1 A-type Proteins 0.000 description 1
- 101710170464 Nuclear factor 1 B-type Proteins 0.000 description 1
- 101710113455 Nuclear factor 1 C-type Proteins 0.000 description 1
- 101710140810 Nuclear factor 1 X-type Proteins 0.000 description 1
- 102100037226 Nuclear receptor coactivator 2 Human genes 0.000 description 1
- 102100039019 Nuclear receptor subfamily 0 group B member 1 Human genes 0.000 description 1
- 102100023170 Nuclear receptor subfamily 1 group D member 1 Human genes 0.000 description 1
- 102100023171 Nuclear receptor subfamily 1 group D member 2 Human genes 0.000 description 1
- 102100038512 Nuclear receptor subfamily 1 group I member 3 Human genes 0.000 description 1
- 102100028470 Nuclear receptor subfamily 2 group C member 1 Human genes 0.000 description 1
- 102100029528 Nuclear receptor subfamily 2 group F member 6 Human genes 0.000 description 1
- 102100022676 Nuclear receptor subfamily 4 group A member 2 Human genes 0.000 description 1
- 102100034408 Nuclear transcription factor Y subunit alpha Human genes 0.000 description 1
- 101710115878 Nuclear transcription factor Y subunit alpha Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- JXNORPPTKDEAIZ-QOCRDCMYSA-N O-4''-alpha-D-mannosylqueuosine Chemical compound NC(N1)=NC(N([C@@H]([C@@H]2O)O[C@H](CO)[C@H]2O)C=C2CN[C@H]([C@H]3O)C=C[C@@H]3O[C@H]([C@H]([C@H]3O)O)O[C@H](CO)[C@H]3O)=C2C1=O JXNORPPTKDEAIZ-QOCRDCMYSA-N 0.000 description 1
- XMIFBEZRFMTGRL-TURQNECASA-N OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)n1cc(CNCCS(O)(=O)=O)c(=O)[nH]c1=S Chemical compound OC[C@H]1O[C@H]([C@H](O)[C@@H]1O)n1cc(CNCCS(O)(=O)=O)c(=O)[nH]c1=S XMIFBEZRFMTGRL-TURQNECASA-N 0.000 description 1
- 229910004679 ONO2 Inorganic materials 0.000 description 1
- 101150092239 OTX2 gene Proteins 0.000 description 1
- 102100021079 Ornithine decarboxylase Human genes 0.000 description 1
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 1
- 101150041192 Otx1 gene Proteins 0.000 description 1
- 102100034819 PDZ and LIM domain protein 1 Human genes 0.000 description 1
- 102100030476 POU domain class 2-associating factor 1 Human genes 0.000 description 1
- 101710114665 POU domain class 2-associating factor 1 Proteins 0.000 description 1
- 102100035593 POU domain, class 2, transcription factor 1 Human genes 0.000 description 1
- 101710084414 POU domain, class 2, transcription factor 1 Proteins 0.000 description 1
- 102100035591 POU domain, class 2, transcription factor 2 Human genes 0.000 description 1
- 101710084411 POU domain, class 2, transcription factor 2 Proteins 0.000 description 1
- 102100026450 POU domain, class 3, transcription factor 4 Human genes 0.000 description 1
- 101710133389 POU domain, class 3, transcription factor 4 Proteins 0.000 description 1
- 102100035394 POU domain, class 4, transcription factor 2 Human genes 0.000 description 1
- 101150054854 POU1F1 gene Proteins 0.000 description 1
- 102000023984 PPAR alpha Human genes 0.000 description 1
- 102000000536 PPAR gamma Human genes 0.000 description 1
- 108010044210 PPAR-beta Proteins 0.000 description 1
- 108091008768 PPARγ1 Proteins 0.000 description 1
- 108091008767 PPARγ2 Proteins 0.000 description 1
- 102100041030 Pancreas/duodenum homeobox protein 1 Human genes 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 101100312945 Pasteurella multocida (strain Pm70) talA gene Proteins 0.000 description 1
- 101100536300 Pasteurella multocida (strain Pm70) talB gene Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102100020739 Peptidyl-prolyl cis-trans isomerase FKBP4 Human genes 0.000 description 1
- 108010043958 Peptoids Proteins 0.000 description 1
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 description 1
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical class OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 description 1
- 102100029533 Photoreceptor-specific nuclear receptor Human genes 0.000 description 1
- 102100030345 Pituitary homeobox 1 Human genes 0.000 description 1
- 102100036090 Pituitary homeobox 2 Human genes 0.000 description 1
- 102100036088 Pituitary homeobox 3 Human genes 0.000 description 1
- 102100034960 Poly(rC)-binding protein 1 Human genes 0.000 description 1
- 101710089655 Poly(rC)-binding protein 1 Proteins 0.000 description 1
- 239000004952 Polyamide Substances 0.000 description 1
- 102100030702 Polycomb protein SUZ12 Human genes 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 108010012271 Positive Transcriptional Elongation Factor B Proteins 0.000 description 1
- 102000019014 Positive Transcriptional Elongation Factor B Human genes 0.000 description 1
- 208000002389 Pouchitis Diseases 0.000 description 1
- 102100025513 Prefoldin subunit 5 Human genes 0.000 description 1
- 101710145525 Probable cinnamyl alcohol dehydrogenase Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108700003766 Promyelocytic Leukemia Zinc Finger Proteins 0.000 description 1
- 108700017836 Prophet of Pit-1 Proteins 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Chemical class CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 1
- 101000896142 Prorocentrum triestinum Blooming-related protein 2 Proteins 0.000 description 1
- 102100025198 Protein DBF4 homolog A Human genes 0.000 description 1
- 102100027171 Protein SET Human genes 0.000 description 1
- 101710148582 Protein SET Proteins 0.000 description 1
- 102100028588 Protein ZNRD2 Human genes 0.000 description 1
- 102100037925 Prothymosin alpha Human genes 0.000 description 1
- 102000052575 Proto-Oncogene Human genes 0.000 description 1
- 108700020978 Proto-Oncogene Proteins 0.000 description 1
- 101001098058 Protobothrops flavoviridis Basic phospholipase A2 BP-I Proteins 0.000 description 1
- 101001133899 Protobothrops flavoviridis Basic phospholipase A2 BP-II Proteins 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 1
- 102100021168 Putative double homeobox protein 3 Human genes 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- 108091008730 RAR-related orphan receptors β Proteins 0.000 description 1
- 108091008773 RAR-related orphan receptors γ Proteins 0.000 description 1
- 108091008103 RNA aptamers Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 102000044126 RNA-Binding Proteins Human genes 0.000 description 1
- 101710105008 RNA-binding protein Proteins 0.000 description 1
- 208000037340 Rare genetic disease Diseases 0.000 description 1
- 102100023544 Ras-responsive element-binding protein 1 Human genes 0.000 description 1
- 101710132554 Ras-responsive element-binding protein 1 Proteins 0.000 description 1
- 101001041232 Rattus norvegicus Ornithine decarboxylase Proteins 0.000 description 1
- 101100533701 Rattus norvegicus Smad1 gene Proteins 0.000 description 1
- 101100431670 Rattus norvegicus Ybx3 gene Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010030933 Regulatory Factor X1 Proteins 0.000 description 1
- 108091027981 Response element Proteins 0.000 description 1
- 108050002592 Retinoblastoma-like protein 1 Proteins 0.000 description 1
- 102100035178 Retinoic acid receptor RXR-alpha Human genes 0.000 description 1
- 102100034253 Retinoic acid receptor RXR-beta Human genes 0.000 description 1
- 102100033909 Retinoic acid receptor beta Human genes 0.000 description 1
- 102100033912 Retinoic acid receptor gamma Human genes 0.000 description 1
- 108091008770 Rev-ErbAß Proteins 0.000 description 1
- 102100030676 Rho GTPase-activating protein 35 Human genes 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 102100022092 Ribosomal oxygenase 2 Human genes 0.000 description 1
- 102100027482 Ribosome production factor 1 Human genes 0.000 description 1
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 1
- 102100025373 Runt-related transcription factor 1 Human genes 0.000 description 1
- 102100025368 Runt-related transcription factor 2 Human genes 0.000 description 1
- 102100025369 Runt-related transcription factor 3 Human genes 0.000 description 1
- 102100027160 RuvB-like 1 Human genes 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 102100021782 SH3 domain-containing protein 19 Human genes 0.000 description 1
- 101700032040 SMAD1 Proteins 0.000 description 1
- 102000004265 STAT2 Transcription Factor Human genes 0.000 description 1
- 108010081691 STAT2 Transcription Factor Proteins 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 102000005886 STAT4 Transcription Factor Human genes 0.000 description 1
- 108010019992 STAT4 Transcription Factor Proteins 0.000 description 1
- 206010039491 Sarcoma Diseases 0.000 description 1
- 240000005499 Sasa Species 0.000 description 1
- 101100528938 Schizosaccharomyces pombe (strain 972 / ATCC 24843) ker1 gene Proteins 0.000 description 1
- 101100368917 Schizosaccharomyces pombe (strain 972 / ATCC 24843) taz1 gene Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101100174184 Serratia marcescens fosA gene Proteins 0.000 description 1
- 108010042291 Serum Response Factor Proteins 0.000 description 1
- 102100022056 Serum response factor Human genes 0.000 description 1
- 102100022978 Sex-determining region Y protein Human genes 0.000 description 1
- 102100029904 Signal transducer and activator of transcription 1-alpha/beta Human genes 0.000 description 1
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 1
- 108091027568 Single-stranded nucleotide Proteins 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 101150117830 Sox5 gene Proteins 0.000 description 1
- 208000000102 Squamous Cell Carcinoma of Head and Neck Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 102100036832 Steroid hormone receptor ERR1 Human genes 0.000 description 1
- 102100036831 Steroid hormone receptor ERR2 Human genes 0.000 description 1
- 102000008078 Sterol Regulatory Element Binding Protein 1 Human genes 0.000 description 1
- 108010074438 Sterol Regulatory Element Binding Protein 2 Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 description 1
- 108010029625 T-Box Domain Protein 2 Proteins 0.000 description 1
- 102100038721 T-box transcription factor TBX2 Human genes 0.000 description 1
- 102100024754 T-box transcription factor TBX4 Human genes 0.000 description 1
- 102100033111 T-cell leukemia homeobox protein 1 Human genes 0.000 description 1
- 102100032568 T-cell leukemia homeobox protein 3 Human genes 0.000 description 1
- 102100028866 TATA element modulatory factor Human genes 0.000 description 1
- 101710136628 TATA element modulatory factor Proteins 0.000 description 1
- 102100028639 TATA-binding protein-associated factor 172 Human genes 0.000 description 1
- 239000008051 TBE buffer Substances 0.000 description 1
- 238000012338 Therapeutic targeting Methods 0.000 description 1
- 101710088547 Thyroid transcription factor 1 Proteins 0.000 description 1
- 102100031224 Tonsoku-like protein Human genes 0.000 description 1
- 101710169241 Tonsoku-like protein Proteins 0.000 description 1
- 230000010632 Transcription Factor Activity Effects 0.000 description 1
- 108010083262 Transcription Factor TFIIA Proteins 0.000 description 1
- 108010083268 Transcription Factor TFIID Proteins 0.000 description 1
- 102100038997 Transcription elongation factor SPT4 Human genes 0.000 description 1
- 102100030402 Transcription elongation factor SPT5 Human genes 0.000 description 1
- 108050005285 Transcription factor 7-like 1 Proteins 0.000 description 1
- 101710138750 Transcription factor E2F1 Proteins 0.000 description 1
- 101710138752 Transcription factor E2F3 Proteins 0.000 description 1
- 108050002596 Transcription factor E2F5 Proteins 0.000 description 1
- 108050006733 Transcription factor E2F6 Proteins 0.000 description 1
- 102100028336 Transcription factor HIVEP3 Human genes 0.000 description 1
- 101710177551 Transcription factor HIVEP3 Proteins 0.000 description 1
- 102100034738 Transcription factor LBX1 Human genes 0.000 description 1
- 102100027654 Transcription factor PU.1 Human genes 0.000 description 1
- 102100022821 Transcription factor RFX3 Human genes 0.000 description 1
- 102000004408 Transcription factor TFIIB Human genes 0.000 description 1
- 108090000941 Transcription factor TFIIB Proteins 0.000 description 1
- 101710145409 Transcription initiation factor IIA subunit 2 Proteins 0.000 description 1
- 101710165271 Transcription initiation factor IIF subunit alpha Proteins 0.000 description 1
- 101710156229 Transcription initiation factor IIF subunit beta Proteins 0.000 description 1
- 108050004072 Transcription initiation factor TFIID subunit 1 Proteins 0.000 description 1
- 102100036677 Transcription initiation factor TFIID subunit 10 Human genes 0.000 description 1
- 101710185107 Transcription initiation factor TFIID subunit 10 Proteins 0.000 description 1
- 102100036676 Transcription initiation factor TFIID subunit 11 Human genes 0.000 description 1
- 101710185106 Transcription initiation factor TFIID subunit 11 Proteins 0.000 description 1
- 102100025941 Transcription initiation factor TFIID subunit 13 Human genes 0.000 description 1
- 101710185097 Transcription initiation factor TFIID subunit 13 Proteins 0.000 description 1
- 102100030833 Transcription initiation factor TFIID subunit 4 Human genes 0.000 description 1
- 102100021230 Transcription initiation factor TFIID subunit 5 Human genes 0.000 description 1
- 101710104808 Transcription initiation factor TFIID subunit 5 Proteins 0.000 description 1
- 102100034748 Transcription initiation factor TFIID subunit 7 Human genes 0.000 description 1
- 101710104820 Transcription initiation factor TFIID subunit 7 Proteins 0.000 description 1
- 101710159262 Transcription termination factor 1 Proteins 0.000 description 1
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 1
- 102100035146 Transcriptional enhancer factor TEF-4 Human genes 0.000 description 1
- 101710152982 Transcriptional enhancer factor TEF-4 Proteins 0.000 description 1
- 102100027671 Transcriptional repressor CTCF Human genes 0.000 description 1
- 102100039362 Transducin-like enhancer protein 1 Human genes 0.000 description 1
- 108050008367 Transmembrane emp24 domain-containing protein 7 Proteins 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- OKKRPWIIYQTPQF-UHFFFAOYSA-N Trimethylolpropane trimethacrylate Chemical compound CC(=C)C(=O)OCC(CC)(COC(=O)C(C)=C)COC(=O)C(C)=C OKKRPWIIYQTPQF-UHFFFAOYSA-N 0.000 description 1
- 102000015098 Tumor Suppressor Protein p53 Human genes 0.000 description 1
- 108010078814 Tumor Suppressor Protein p53 Proteins 0.000 description 1
- 208000035896 Twin-reversed arterial perfusion sequence Diseases 0.000 description 1
- 206010045261 Type IIa hyperlipidaemia Diseases 0.000 description 1
- 102100021575 Tyrosine-protein kinase BAZ1B Human genes 0.000 description 1
- 102100040065 Upstream-binding protein 1 Human genes 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 102100028983 Vascular endothelial zinc finger 1 Human genes 0.000 description 1
- JCZSFCLRSONYLH-UHFFFAOYSA-N Wyosine Natural products N=1C(C)=CN(C(C=2N=C3)=O)C=1N(C)C=2N3C1OC(CO)C(O)C1O JCZSFCLRSONYLH-UHFFFAOYSA-N 0.000 description 1
- 108010035430 X-Box Binding Protein 1 Proteins 0.000 description 1
- YXNIEZJFCGTDKV-UHFFFAOYSA-N X-Nucleosid Natural products O=C1N(CCC(N)C(O)=O)C(=O)C=CN1C1C(O)C(O)C(CO)O1 YXNIEZJFCGTDKV-UHFFFAOYSA-N 0.000 description 1
- 102100038151 X-box-binding protein 1 Human genes 0.000 description 1
- 101000954901 Xenopus laevis Ornithine decarboxylase 1 Proteins 0.000 description 1
- 101100351021 Xenopus laevis pax5 gene Proteins 0.000 description 1
- 108700031763 Xeroderma Pigmentosum Group D Proteins 0.000 description 1
- 102100027644 YY1-associated factor 2 Human genes 0.000 description 1
- 102100023405 Zinc finger X-chromosomal protein Human genes 0.000 description 1
- 102100040314 Zinc finger and BTB domain-containing protein 16 Human genes 0.000 description 1
- 102100040761 Zinc finger and BTB domain-containing protein 17 Human genes 0.000 description 1
- 102100025396 Zinc finger and BTB domain-containing protein 6 Human genes 0.000 description 1
- 102100026583 Zinc finger and SCAN domain-containing protein 26 Human genes 0.000 description 1
- 102100040812 Zinc finger protein 174 Human genes 0.000 description 1
- 102100023492 Zinc finger protein ZIC 2 Human genes 0.000 description 1
- 102100025105 Zinc fingers and homeoboxes protein 1 Human genes 0.000 description 1
- TVGUROHJABCRTB-MHJQXXNXSA-N [(2r,3s,4r,5s)-5-[(2r,3r,4r,5r)-2-(2-amino-6-oxo-3h-purin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl]oxy-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate Chemical compound O([C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C=NC=2C(=O)N=C(NC=21)N)[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O TVGUROHJABCRTB-MHJQXXNXSA-N 0.000 description 1
- HIHOWBSBBDRPDW-PTHRTHQKSA-N [(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-yl] n-[2-(dimethylamino)ethyl]carbamate Chemical compound C1C=C2C[C@@H](OC(=O)NCCN(C)C)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HIHOWBSBBDRPDW-PTHRTHQKSA-N 0.000 description 1
- HMNZFMSWFCAGGW-XPWSMXQVSA-N [3-[hydroxy(2-hydroxyethoxy)phosphoryl]oxy-2-[(e)-octadec-9-enoyl]oxypropyl] (e)-octadec-9-enoate Chemical compound CCCCCCCC\C=C\CCCCCCCC(=O)OCC(COP(O)(=O)OCCO)OC(=O)CCCCCCC\C=C\CCCCCCCC HMNZFMSWFCAGGW-XPWSMXQVSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 1
- 229960005305 adenosine Drugs 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 208000026594 alcoholic fatty liver disease Diseases 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 125000002723 alicyclic group Chemical group 0.000 description 1
- 125000002009 alkene group Chemical group 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 125000005083 alkoxyalkoxy group Chemical group 0.000 description 1
- 125000002877 alkyl aryl group Chemical group 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000005122 aminoalkylamino group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- PYKYMHQGRFAEBM-UHFFFAOYSA-N anthraquinone Natural products CCC(=O)c1c(O)c2C(=O)C3C(C=CC=C3O)C(=O)c2cc1CC(=O)OC PYKYMHQGRFAEBM-UHFFFAOYSA-N 0.000 description 1
- 150000004056 anthraquinones Chemical class 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- PEMQXWCOMFJRLS-RPKMEZRRSA-N archaeosine Chemical compound C1=2NC(N)=NC(=O)C=2C(C(=N)N)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O PEMQXWCOMFJRLS-RPKMEZRRSA-N 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 125000003710 aryl alkyl group Chemical group 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 208000006673 asthma Diseases 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 238000005284 basis set Methods 0.000 description 1
- 102000055102 bcl-2-Associated X Human genes 0.000 description 1
- 108700000707 bcl-2-Associated X Proteins 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N benzo-alpha-pyrone Natural products C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 125000002619 bicyclic group Chemical group 0.000 description 1
- 230000008275 binding mechanism Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 201000000053 blastoma Diseases 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000000133 brain stem Anatomy 0.000 description 1
- 150000003842 bromide salts Chemical class 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 102100035161 c-Myc-binding protein Human genes 0.000 description 1
- 101710193923 c-Myc-binding protein Proteins 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 235000011132 calcium sulphate Nutrition 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 125000001369 canonical nucleoside group Chemical group 0.000 description 1
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 1
- 101150073031 cdk2 gene Proteins 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 230000033077 cellular process Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 206010008129 cerebral palsy Diseases 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001841 cholesterols Chemical class 0.000 description 1
- 238000002983 circular dichroism Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 101150118300 cos gene Proteins 0.000 description 1
- 235000001671 coumarin Nutrition 0.000 description 1
- 125000000332 coumarinyl group Chemical class O1C(=O)C(=CC2=CC=CC=C12)* 0.000 description 1
- 125000001995 cyclobutyl group Chemical group [H]C1([H])C([H])([H])C([H])(*)C1([H])[H] 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- ZPTBLXKRQACLCR-XVFCMESISA-N dihydrouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)CC1 ZPTBLXKRQACLCR-XVFCMESISA-N 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- WWJJVKAEQGGYHJ-UHFFFAOYSA-N dimethyl thiophosphate Chemical compound COP(O)(=S)OC WWJJVKAEQGGYHJ-UHFFFAOYSA-N 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-N dithiophosphoric acid Chemical class OP(O)(S)=S NAGJZTKCGNOGPW-UHFFFAOYSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 108700002489 ebola virus VP30 Proteins 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 201000008184 embryoma Diseases 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- RRCFLRBBBFZLSB-XIFYLAFSSA-N epoxyqueuosine Chemical compound C1=C(CN[C@@H]2[C@H]([C@@H](O)[C@@H]3O[C@@H]32)O)C=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O RRCFLRBBBFZLSB-XIFYLAFSSA-N 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 201000001386 familial hypercholesterolemia Diseases 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 229940014144 folate Drugs 0.000 description 1
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 1
- 235000019152 folic acid Nutrition 0.000 description 1
- 239000011724 folic acid Substances 0.000 description 1
- 101150078861 fos gene Proteins 0.000 description 1
- 238000002825 functional assay Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 230000005021 gait Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 230000030279 gene silencing Effects 0.000 description 1
- 238000012226 gene silencing method Methods 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 125000000592 heterocycloalkyl group Chemical group 0.000 description 1
- 108010021685 homeobox protein HOXA13 Proteins 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 101150118036 hoxa9a gene Proteins 0.000 description 1
- 101150019766 hoxa9b gene Proteins 0.000 description 1
- 102000053413 human GT-IC Human genes 0.000 description 1
- 108700042383 human GT-IC Proteins 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 229920001600 hydrophobic polymer Polymers 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 208000006575 hypertriglyceridemia Diseases 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 210000004263 induced pluripotent stem cell Anatomy 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 238000003331 infrared imaging Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 239000000138 intercalating agent Substances 0.000 description 1
- 108010051621 interferon regulatory factor-8 Proteins 0.000 description 1
- 238000000185 intracerebroventricular administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 208000017476 juvenile neuronal ceroid lipofuscinosis Diseases 0.000 description 1
- 108091008792 l-Myc Proteins 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 108090000865 liver X receptors Proteins 0.000 description 1
- 102000004311 liver X receptors Human genes 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 201000005249 lung adenocarcinoma Diseases 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 150000002690 malonic acid derivatives Chemical class 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- HLZXTFWTDIBXDF-UHFFFAOYSA-N mcm5sU Natural products COC(=O)Cc1cn(C2OC(CO)C(O)C2O)c(=S)[nH]c1=O HLZXTFWTDIBXDF-UHFFFAOYSA-N 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000007721 medicinal effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 101150029117 meox2 gene Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- GWKIZNPISGBQGY-GNLDREGESA-N methyl (2S)-4-[4,6-dimethyl-9-oxo-3-[(2R,3R,4S,5R)-2,3,4-trihydroxy-5-(hydroxymethyl)oxolan-2-yl]imidazo[1,2-a]purin-7-yl]-2-(methoxycarbonylamino)butanoate Chemical class O[C@@]1([C@H](O)[C@H](O)[C@@H](CO)O1)N1C=NC=2C(=O)N3C(CC[C@@H](C(=O)OC)NC(=O)OC)=C(C)N=C3N(C)C21 GWKIZNPISGBQGY-GNLDREGESA-N 0.000 description 1
- JNVLKTZUCGRYNN-LQGIRWEJSA-N methyl 2-[1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-2,4-dioxopyrimidin-5-yl]-2-hydroxyacetate Chemical compound O=C1NC(=O)C(C(O)C(=O)OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 JNVLKTZUCGRYNN-LQGIRWEJSA-N 0.000 description 1
- WCNMEQDMUYVWMJ-UHFFFAOYSA-N methyl 4-[3-[3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-4,6-dimethyl-9-oxoimidazo[1,2-a]purin-7-yl]-3-hydroperoxy-2-(methoxycarbonylamino)butanoate Chemical compound C1=NC=2C(=O)N3C(CC(C(NC(=O)OC)C(=O)OC)OO)=C(C)N=C3N(C)C=2N1C1OC(CO)C(O)C1O WCNMEQDMUYVWMJ-UHFFFAOYSA-N 0.000 description 1
- WZRYXYRWFAPPBJ-PNHWDRBUSA-N methyl uridin-5-yloxyacetate Chemical compound O=C1NC(=O)C(OCC(=O)OC)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 WZRYXYRWFAPPBJ-PNHWDRBUSA-N 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical compound CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 101150084874 mimG gene Proteins 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 101150094258 mxi1 gene Proteins 0.000 description 1
- 108010084677 myogenic factor 6 Proteins 0.000 description 1
- 108091008800 n-Myc Proteins 0.000 description 1
- CYDFBLGNJUNSCC-QCNRFFRDSA-N n-[1-[(2r,3r,4r,5r)-4-hydroxy-5-(hydroxymethyl)-3-methoxyoxolan-2-yl]-2-oxopyrimidin-4-yl]acetamide Chemical compound CO[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)N=C(NC(C)=O)C=C1 CYDFBLGNJUNSCC-QCNRFFRDSA-N 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 201000011216 nasopharynx carcinoma Diseases 0.000 description 1
- 230000002232 neuromuscular Effects 0.000 description 1
- 208000018360 neuromuscular disease Diseases 0.000 description 1
- 201000007607 neuronal ceroid lipofuscinosis 3 Diseases 0.000 description 1
- 125000001893 nitrooxy group Chemical group [O-][N+](=O)O* 0.000 description 1
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 1
- 101150083701 npm1 gene Proteins 0.000 description 1
- 108010010765 nuclear factor-jun Proteins 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 238000011903 nutritional therapy Methods 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000007764 o/w emulsion Substances 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 229940124276 oligodeoxyribonucleotide Drugs 0.000 description 1
- 238000002515 oligonucleotide synthesis Methods 0.000 description 1
- 108700030515 omomyc Proteins 0.000 description 1
- 108091008819 oncoproteins Proteins 0.000 description 1
- 201000002740 oral squamous cell carcinoma Diseases 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 125000001181 organosilyl group Chemical group [SiH3]* 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 101800000607 p15 Proteins 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 101150098999 pax8 gene Proteins 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 108091008725 peroxisome proliferator-activated receptors alpha Proteins 0.000 description 1
- 229950000688 phenothiazine Drugs 0.000 description 1
- 150000002991 phenoxazines Chemical class 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- UEZVMMHDMIWARA-UHFFFAOYSA-M phosphonate Chemical compound [O-]P(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-M 0.000 description 1
- 150000008298 phosphoramidates Chemical class 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 238000012913 prioritisation Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 230000004952 protein activity Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 108010008929 proto-oncogene protein Spi-1 Proteins 0.000 description 1
- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- UBQKCCHYAOITMY-UHFFFAOYSA-N pyridin-2-ol Chemical compound OC1=CC=CC=N1 UBQKCCHYAOITMY-UHFFFAOYSA-N 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- RXTQGIIIYVEHBN-UHFFFAOYSA-N pyrimido[4,5-b]indol-2-one Chemical compound C1=CC=CC2=NC3=NC(=O)N=CC3=C21 RXTQGIIIYVEHBN-UHFFFAOYSA-N 0.000 description 1
- 238000003077 quantum chemistry computational method Methods 0.000 description 1
- QQXQGKSPIMGUIZ-AEZJAUAXSA-N queuosine Chemical compound C1=2C(=O)NC(N)=NC=2N([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)C=C1CN[C@H]1C=C[C@H](O)[C@@H]1O QQXQGKSPIMGUIZ-AEZJAUAXSA-N 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 125000006853 reporter group Chemical group 0.000 description 1
- 230000001718 repressive effect Effects 0.000 description 1
- 210000004994 reproductive system Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108010059301 retinoic acid receptor gamma Proteins 0.000 description 1
- 108091008761 retinoic acid receptors β Proteins 0.000 description 1
- 108091008760 retinoic acid receptors γ Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 101150118809 rox gene Proteins 0.000 description 1
- RHFUOMFWUGWKKO-UHFFFAOYSA-N s2C Natural products S=C1N=C(N)C=CN1C1C(O)C(O)C(CO)O1 RHFUOMFWUGWKKO-UHFFFAOYSA-N 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000013077 scoring method Methods 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 102100024840 snRNA-activating protein complex subunit 1 Human genes 0.000 description 1
- 102100024838 snRNA-activating protein complex subunit 2 Human genes 0.000 description 1
- 102100022779 snRNA-activating protein complex subunit 3 Human genes 0.000 description 1
- 102100022780 snRNA-activating protein complex subunit 4 Human genes 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 208000002320 spinal muscular atrophy Diseases 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 125000002345 steroid group Chemical group 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical group NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- 150000003456 sulfonamides Chemical group 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 150000003457 sulfones Chemical group 0.000 description 1
- 150000003462 sulfoxides Chemical class 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000759 toxicological effect Toxicity 0.000 description 1
- 108010072897 transcription factor Brn-2 Proteins 0.000 description 1
- 108010014678 transcription factor TFIIF Proteins 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
- 125000002023 trifluoromethyl group Chemical group FC(F)(F)* 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
- 125000004417 unsaturated alkyl group Chemical group 0.000 description 1
- RVCNQQGZJWVLIP-VPCXQMTMSA-N uridin-5-yloxyacetic acid Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(OCC(O)=O)=C1 RVCNQQGZJWVLIP-VPCXQMTMSA-N 0.000 description 1
- YIZYCHKPHCPKHZ-UHFFFAOYSA-N uridine-5-acetic acid methyl ester Natural products COC(=O)Cc1cn(C2OC(CO)C(O)C2O)c(=O)[nH]c1=O YIZYCHKPHCPKHZ-UHFFFAOYSA-N 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000004393 visual impairment Effects 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- QAOHCFGKCWTBGC-QHOAOGIMSA-N wybutosine Chemical compound C1=NC=2C(=O)N3C(CC[C@H](NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O QAOHCFGKCWTBGC-QHOAOGIMSA-N 0.000 description 1
- QAOHCFGKCWTBGC-UHFFFAOYSA-N wybutosine Natural products C1=NC=2C(=O)N3C(CCC(NC(=O)OC)C(=O)OC)=C(C)N=C3N(C)C=2N1C1OC(CO)C(O)C1O QAOHCFGKCWTBGC-UHFFFAOYSA-N 0.000 description 1
- JCZSFCLRSONYLH-QYVSTXNMSA-N wyosin Chemical compound N=1C(C)=CN(C(C=2N=C3)=O)C=1N(C)C=2N3[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O JCZSFCLRSONYLH-QYVSTXNMSA-N 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
- 238000012447 xenograft mouse model Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/13—Decoys
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/50—Physical structure
- C12N2310/53—Physical structure partially self-complementary or closed
- C12N2310/531—Stem-loop; Hairpin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
Definitions
- the present disclosure relates to the field of oligonucleotides. More particularly, the present disclosure relates to hairpin oligonucleotides for the specific binding and inhibition of target proteins, such as transcription factors. Additionally, this disclosure relates to methods of using such oligonucleotides in preventing or treating diseases, disorders or conditions associated with such target proteins.
- Nucleic acid binding proteins such as transcription factors
- transcription factors can play a role at various points in cell signalling pathways, so as to modulate many normal cellular processes, such as cell growth and proliferation, metabolism, apoptosis, immune responses, and differentiation. Given these cellular effects, their activity is often found to be deregulated in disease, such as cancer. As such, targeting this class of proteins is a major focus of interest, but the structural disorder and lack of binding pockets have made the rational design of small molecule inhibitors for transcription factors challenging. Accordingly, there remains a need for new inhibitors of nucleic acid binding proteins, and particularly those that can specifically target transcription factor activity.
- the present disclosure is based on the surprising finding that a hairpin oligonucleotide that includes a consensus cMyc binding site in its stem region can specifically bind to this transcription factor with high affinity and inhibit its proliferative effects in cancer cells. Such an inhibition strategy may be applied to other transcription factors that bind to specific nucleic acid sequences, structures or motifs with high affinity.
- the present disclosure provides a hairpin oligonucleotide for inhibiting a target protein, the hairpin oligonucleotide comprising a loop region and a stem region, wherein the stem region comprises a binding site for the target protein.
- the target protein is a transcription factor, such as an E-box transcription factor.
- the transcription factor is selected from the group consisting of Myc, Myc/Max, Fos/Jun, HIF1 ⁇ / ⁇ , MITF, MyoD, HES family, Hey family, ID1/2/3, E2 family, Twist, AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATF1, ATF2, ATF4, ATF5, ATF6, ATF7, ATOH1, ATOH7, ATOH8, BACH1, BACH2, BATF, BATF2, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, CREB1, CREB3, CREB3L1, CREB3L2, CREB3L3, CREB3L4, CREB5, CREBL1, CREM, E4BP4, EPAS1, FERD3L, FIGLA, FOSL1, FOSL2, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7,
- the transcription factor is Myc or Myc/Max.
- the binding site comprises, consists of or consists essentially of the nucleic acid sequence of 5'-CAC[GA]TG-3', 5'-CAC[GA]UG-3' or a fragment, variant or derivative thereof.
- the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-GAGCACGUGGUU-3' (SEQ ID NO: 3) or a fragment, variant or derivative thereof and wherein one or more of the U nucleotides thereof may be a T.
- the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleotide sequence of SEQ ID NO: 1 or a fragment, variant or derivative thereof.
- the stem region comprises a sense strand and an antisense strand, each strand having 5 to 50 nucleotides.
- the hairpin oligonucleotide comprises one or a plurality of phosphorothioate internucleotide linkages.
- the hairpin oligonucleotide comprises one or a plurality of modified nucleotides.
- the one or plurality of modified nucleotides comprise a modification selected from the group consisting of LNA, HNA, CeNA, 2’-methoxyethyl, 2’-O-alkyl, 2’-O-allyl, 2’- C- allyl, 2’-fluoro, 2’-deoxy, and combinations thereof.
- one or more of the modified nucleotides are modified with 2’-OCH 3 .
- the present disclosure provides a pharmaceutical composition comprising the hairpin oligonucleotide of the first aspect and a pharmaceutically-acceptable carrier, diluent or excipient.
- the present disclosure relates to a method of inhibiting a target protein of a cell, said method including the step of contacting the cell with the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect.
- the present disclosure provides for the use of the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect in the manufacture of a medicament for inhibiting a target protein of a cell.
- the present disclosure resides in a method of preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject, said method including the step of administering to the subject a therapeutically effective amount of the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect to thereby prevent, ameliorate or treat the disease, disorder or condition.
- the present disclosure relates to the use of the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect in the manufacture of a medicament for preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject.
- the disease, disorder or condition suitably is or comprises a cancer.
- the cancer is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, cancer of the brain and nervous system, head and neck cancer, colon cancer, colorectal cancer, gastric cancer, liver cancer, kidney cancer, melanoma, skin carcinoma, lymphoid cancer, myelomonocytic cancer, pancreatic cancer, pituitary cancer, bone cancer and soft tissue cancer.
- the cancer is or comprises breast cancer.
- the present disclosure provides a method for selecting a hairpin oligonucleotide for inhibiting a target protein, said method including the steps of: (a) producing one or more candidate hairpin oligonucleotides comprising a loop region and a stem region, wherein the stem region comprises a binding site for the target protein; (b) testing the ability of the one or more candidate hairpin oligonucleotides to inhibit the target protein, and (c) selecting a hairpin oligonucleotide which binds and/or inhibits the target protein.
- the present disclosure relates to a hairpin oligonucleotide designed, selected or identified by the method of the seventh aspect.
- Bases are labelled according to the sequence's 5' to 3' position.
- B Diagrammatic secondary structure representation of DRpinMYC from MC-Fold composed of twelve Watson-Crick base pairs with a four-base poly-Adenosine loop and blunt ends.
- C MC-Fold generated dot-bracket notation of secondary structure of DRpinMYC. Scoring indicates a highly stable structure.
- Figure 2 Binding of DRpin to cMyc:Max dimer.
- Left panel Graph of densitometry of cMyc binding to DRpin-Myc
- Right panel Graph of densitometry of the Myc:Max dimer binding to DRpin-Myc.
- NegC is an oligonucleotide of the same chemistry, but lacking the Myc consensus sequence (random sequence).
- Y axis is relative DRpin-Myc bound by Myc or Myc:Max.
- X axis is nM of Myc or Myc:Max.
- Figure 3 A diagram depicting the proposed binding of DRpinMyc with cMyc:Max. Modelling is consistent with the EMSA assays. It suggests the self folding RNA DRpin-Myc molecule can be bound by the cMyc:Max heterodimer.
- Figure 4 DRpinMyc was used to treat a number of breast cancer cell lines.
- Figure 6 Cell cycle analysis was performed by flow cytometry using the PI stain. It indicated that like siRNA deletion of cMyc that the cell arrested at different stages of the cell cycle. A represents a 48 hour time point and B a 120 hour time points. The later time points showed that cells were beginning to die.
- Figure 7 Venn diagram representation of DE genes (fold change beyond +1 and Padj ⁇ 0.05) across all contrasts.
- Figure 8 Heatmap of upregulated transcription factors: Shown are the significantly upregulated transcription factors. The colour scale bar shows z-score values after z-score row normalization. Heatmap was generated using pheatmap package from R.
- Figure 9 Heatmap of down regulated transcription factors: Shown are the significantly down regulated transcription factors.
- the colour scale bar shows z-score values after z-score row normalization.
- Heatmap was generated using pheatmap package from R.
- Figure 10 DRpin-Myc significantly inhibited TNBC PDX tumour growth.
- Figure 11 DRpin-Myc significantly inhibited tumour growth in an MDA-MB-231-based xenograft model.
- Figure 12 MM/PBSA thermodynamic cycle. MM/PBSA thermodynamic cycle of protein in green and ligand in yellow. Adapted from the Amber Tools 16 manual.
- Figure 13 Top clusters of oligonucleotides and the MYC: MAX complex. The top representative cluster of each system, derived from 2 ⁇ s simulations performed in triplicate. Each system represents a total 6 ⁇ s.
- Myc and Max bHLH/LZ domains are indicated by purple and pink ribbon structures, respectively.
- a green and yellow ladder structure indicates the DNA duplex.
- a green ladder structure alone indicates DRpinMYC stereoisomers.
- the red ladder structure indicates the E-box duplex in each system. Representations of top clusters proportion, cluster RMSD and buried SASA between the complex and DRpinMYC are provided below.
- Figure 14 MM/GBSA energy decomposition of MYC: MAX systems. Decomposition heatmaps from oligonucleotide, MYC: MAX trajectories. Energys are derived from MM/GBSA calculations and represent 6 ⁇ s total per system.
- SEQ ID NO: 1 Nucleotide sequence of DRpinMyc in Figure 1 SEQ ID NO: 2 Amino acid sequence of cMyc protein SEQ ID NO: 3 Nucleotide sequence of binding site in DRpinMyc SEQ ID NO: 4 Nucleotide sequence of consensus E-box binding site SEQ ID NO: 5 Nucleotide sequence of exemplary E-box binding site #1 SEQ ID NO: 6 Nucleotide sequence of exemplary E-box binding site #2 SEQ ID NO: 7 Nucleotide sequence of exemplary E-box binding site #3 SEQ ID NO: 8 Nucleotide sequence of non-canonical E-box binding site #1 SEQ ID NO: 9 Nucleotide sequence of non-canonical E-box binding site #2 SEQ ID NO: 10 Nucleotide sequence of non-canonical E-box binding site #3 SEQ ID NO: 11 Nucleotide sequence of exemplary E-box binding site #4 SEQ ID NO: 12 Nucleotide sequence of exemplary
- a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens.
- the term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
- hairpin oligonucleotides The inventors has surprisingly shown that hairpin oligonucleotides that incorporate a consensus binding site for a target nucleic acid binding protein can be utilised to bind and functionally deplete the target protein and thereby inhibit a biological activity thereof, such as cell growth or proliferation.
- a hairpin oligonucleotide for inhibiting a target protein comprising a loop region and a stem region, wherein the stem region comprises a binding or recognition site for the target protein.
- oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), wherein the polymer or oligomer of nucleotide monomers contains any combination of nucleobases (referred to in the art and herein as simply as “base”), modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as “internucleotide linkage”). Oligonucleotides can be single-stranded or double-stranded or a combination thereof.
- a single- stranded oligonucleotide can have double-stranded regions and a double-stranded oligonucleotide can have single-stranded regions (such as a microRNA or shRNA).
- the terms “hairpin oligo” or “hairpin oligonucleotide” are used interchangeably herein and refer to a nucleic acid molecule, which has a segment (also known as a “hairpin loop” or “loop region”) that is not hybridized with or complementary to a segment of the same hairpin oligonucleotide, and a double-stranded linear region (also known as a “stem” or “duplex” region) having segments that are complementary to each other in the same molecule.
- the hairpin oligonucleotide suitably comprises a single-stranded loop region that is positioned between a first self-complementary region (e.g., a sense strand) and a second self- complementary region (e.g., an antisense strand) that define, at least in part, the stem region.
- a first self-complementary region e.g., a sense strand
- a second self- complementary region e.g., an antisense strand
- the hairpin structure of the oligonucleotides described herein is advantageous, as a double stranded oligo of the same chemistry, once entering cells, would have the propensity to equilibrate between corresponding double stranded and single stranded nucleotide structures.
- a single stranded nucleic acid would further likely not re-anneal to its complementary single stranded nucleic acid due to the dilution factor within a cell.
- a hairpin oligonucleotide that melts, denatures or dissociates will still maintain a localised high concentration of the respective complementary single stranded stem sequences, which would favour reannealing and formation of the double stranded stem region.
- double stranded oligonucleotides that melt, denature or dissociate will have the potential to anneal to homologous RNA and DNA molecules giving the potential for off target activity and/or the mopping up of the single stranded portions of the oligonucleotide preventing them from reannealing to form a double stranded structure.
- the hairpin oligonucleotide provided herein would be less likely to bind/hybridise and/or interact with other off-target nucleic acids, because: (a) the corresponding sequence with the greatest homology is within the same molecule; and (b) the size of the hairpin oligonucleotide reduces its propensity to bind to short complementary nucleic acids.
- Oligonucleotides generally refer to relatively short sequences of nucleotides, typically with twenty or fewer bases (or nucleotide units), but can also be significantly longer, such as up to about 160 to about 200 nucleotides.
- the hairpin oligonucleotide provided herein is at least about 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100 nucleotides, or any range therein, in length as a single stranded molecule.
- the hairpin oligonucleotide is suitably about 3 to about 75 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, or 75 nucleotides, or any range therein) in length as a single stranded molecule.
- nucleotides e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,
- the hairpin oligonucleotide is about 15 to about 56, more particularly about 20 to about 40, even more particularly about 25 to about 35, nucleotides in length as a single stranded molecule. In some examples, the hairpin oligonucleotide is about 28 nucleotides in length as a single stranded molecule.
- the stem region is of sufficient length to incorporate a binding site for the target protein.
- the stem region i.e., the double stranded linear or duplex region
- the stem region is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 nucleotide pairs, or any range therein, in length. More particularly, the stem region can be about 6 to about 20 nucleotide pairs in length. Even more particularly, the stem region can be about 8 to about 15 nucleotide pairs in length. Yet even more particularly, the stem region can be about 10 to about 14 nucleotide pairs in length. In certain examples, the stem region is about 12 nucleotide pairs in length.
- the stem region provided herein is stable (i.e., the self- complementary regions are capable of remaining hybridized together) at approximately 37°C, and unhybridize (i.e., denature) at temperatures greater than 50°C.
- the stem region of the hairpin oligonucleotide has a Gibbs free energy (AG) of unfolding under physiological conditions ranging from about -10 kcal/mol to about -50 kcal/mol (e.g., about - 10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -44, -45, -46
- the stem region provided herein has a Gibbs free energy (AG) of unfolding under physiological conditions in the range of about -20 kcal/mol to about -40 kcal/mol. In various examples, the stem region provided herein has a Gibbs free energy (AG) of unfolding under physiological conditions of at least about -20 kcal/mol, at least about -25 kcal/mol, at least about -30 kcal/mol or at least about -35 kcal/mol.
- the term “loop region” refers to a single-stranded region of more than one nucleotide or modified nucleotide that is not base-paired. Suitably, the loop region is of length sufficient to enable formation of the hairpin structure and base pairing of the stem region.
- the loop region provided herein is at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, or any range therein, in length. More particularly, the loop region can be about 3 to about 10 nucleotides in length. Even more particularly, the loop region can be about 4 to about 6 nucleotides in length. In certain examples, the loop region is about 4 nucleotides in length. It is contemplated that the loop region may include any suitable sequence of nucleotides (e.g., A, C, G, T and/or U). In various examples, the loop region comprises, consists of or consists essentially of a plurality (e.g., 3, 4, 5, 6, 7 etc) of adenine (A) bases.
- A adenine
- the loop region comprises, consists of or consists essentially of a nucleotide sequence of 5'-AAAA-3', or a variant or derivative thereof.
- the hairpin oligonucleotide herein may include single- and/or double-stranded DNA and/or RNA.
- the hairpin oligonucleotide herein e.g., the hairpin oligonucleotide of SEQ ID NO: 1 or a hairpin oligonucleotide comprising a binding site of SEQ ID NOs: 3-32
- the hairpin oligonucleotide herein includes single-stranded and double-stranded DNA.
- the hairpin oligonucleotide herein includes single-stranded and double-stranded RNA.
- DNA includes genomic DNA and cDNA.
- RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA.
- the hairpin oligonucleotide may be a DNA-RNA hybrid.
- a hairpin oligonucleotide of the present disclosure comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base.
- nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto.
- variant hairpin oligonucleotides may include, for example, nucleotide sequences of naturally occurring (e.g., allelic) variants and orthologs (e.g., from a different species) of, for example, a binding site, such as an E-box binding site, inclusive of a consensus sequence thereof.
- nucleic acid “variant” shares a definable nucleotide sequence relationship with a reference nucleic acid sequence (e.g., a hairpin oligonucleotide, a binding/recognition site, SEQ ID NOs:1, 3-32).
- the “variant” nucleic acid may have one or a plurality of nucleic acids of the reference nucleic acid sequence deleted or substituted by different nucleic acids. It is well understood in the art that some nucleic acids of a DNA/RNA-based binding or recognition site may be substituted or deleted without changing (or only having minimal change to) the affinity of the target protein therefor.
- nucleic acid variants share at least 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more particularly at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more particularly at least 90%, 91%, 92%, 93%, 94%, or 95% nucleotide sequence identity with an isolated nucleic acid of the invention (e.g., SEQ ID NOs: 1, 3 to 32). Percent sequence identity may be determined by any method known in the art, such as that described herein.
- sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity.
- a “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence.
- the comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences.
- Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected.
- sequence identity is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, having regard to the extent that sequences are identical over a window of comparison.
- a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
- sequence identity may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA).
- nucleic acid fragments such as oligonucleotide fragments.
- a “fragment” is a segment, domain, portion or region of a nucleic acid, which respectively constitutes less than 100% of the nucleotide sequence.
- a non-limiting example is an amplification product or a primer or probe.
- a nucleic acid fragment may comprise, for example, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80 (inclusive of any range therein) contiguous nucleotides of said nucleic acid (e.g., SEQ ID NO: 1).
- nucleic acid derivatives inclusive of oligonucleotide derivatives. Such oligonucleotide derivatives may include, for example, one or more modifications and/or conjugates as described herein.
- the hairpin oligonucleotides may be conjugated to one or more moieties or groups which enhance the activity, cellular distribution or cellular uptake thereof. These moieties or groups may be covalently bound to functional groups such as primary or secondary hydroxyl groups.
- moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers.
- Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes/labels (e.g., Cy5 dye to determine cellular uptake and/or localisation of the hairpin oligonucleotide).
- a hairpin oligonucleotide of the present disclosure suitably does not hybridize to a target or known RNA to reduce translation thereof.
- the hairpin oligonucleotide does not function as an antisense oligonucleotide (i.e., does not comprise a region that is complementary to at least a portion of a specific mRNA molecule, such as encoding an endogenous polypeptide and capable of interfering with a post-transcriptional event, such as mRNA translation).
- the hairpin oligonucleotide is not, or does not form part of, a stranded oligonucleotide for gene silencing (such as RNA interference; i.e., not a double stranded oligonucleotide for siRNA or shRNA).
- the hairpin oligonucleotide is not, or does not function as, an aptamer oligonucleotide.
- the hairpin oligonucleotides described herein e.g., the hairpin oligonucleotide of SEQ ID NO: 1 or a hairpin oligonucleotide comprising a binding site of SEQ ID NO: 3-32
- an aptamer oligonucleotide e.g., an RNA aptamer or a DNA aptamer.
- the hairpin oligonucleotide described herein may comprise a synthetic oligonucleotide sequence.
- a “synthetic oligonucleotide sequence” refers to an oligonucleotide sequence which lacks a corresponding sequence that occurs naturally.
- a synthetic oligonucleotide sequence is not complementary to a specific RNA molecule or portion thereof, such as one encoding an endogenous polypeptide.
- the synthetic oligonucleotide sequence is suitably not capable of directly interfering with a post-transcriptional event, such as RNA translation.
- the hairpin oligonucleotide is capable of inhibiting or reducing an activity of the target protein.
- the phrase “reduces an activity of the target protein” or the like refers to a hairpin oligonucleotide of the present disclosure reducing the ability of a target protein to exert a biological effect.
- this may relate to their ability to contact and/or bind to a DNA sequence, such as a promoter sequence or an enhancer sequence, and modulate (e.g., promote or inhibit) expression of the relevant gene.
- the activity of the target protein, such as contacting and/or binding a DNA sequence is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of that in the absence of the oligonucleotide.
- Inhibition of an activity of the target protein by the hairpin oligonucleotide may be assessed by any means known in the art. In the context of transcription factors, this may be assessed by measuring a level of expression of a target gene, whose expression may be at least partly modulated by the target protein, such as at an mRNA and/or protein level (i.e., transcription and/or translation). This can be directly or indirectly achieved by measuring the amount of RNA encoded by the target gene and/or the amount of protein translated from an encoding RNA.
- this may be assessed by determining an expression level (e.g., mRNA and/or protein) of one or more markers or mediators of the cell cycle (e.g., E2F1, CDK4, CDC25A, p27, p15), apoptosis (e.g., Bax, Mcl-1, Bcl2), cellular proliferation (e.g., MINA53, ID2, PTMA) and/or cellular metabolism (e.g., CAD, LDHA, ODC-1). Additionally, this can be measured indirectly by assessing a level of activation of cell signalling pathways downstream of the target gene or target protein in question.
- an expression level e.g., mRNA and/or protein
- markers or mediators of the cell cycle e.g., E2F1, CDK4, CDC25A, p27, p15
- apoptosis e.g., Bax, Mcl-1, Bcl2
- cellular proliferation e.g., MINA53, ID
- a level of cMyc signalling may be determined by assessing a level of cell cycle activity, apoptotic activity, cellular proliferation and/or cellular metabolism.
- a hairpin oligonucleotide of the present disclosure will be synthesized in vitro, such as by chemical synthesis (e.g., solid-phase synthesis).
- chemical synthesis e.g., solid-phase synthesis
- modified bases and a modified backbone are not required, they can be expressed in vitro or in vivo in a suitable system, such as by a recombinant virus or cell.
- the hairpin oligonucleotides described herein are isolated.
- isolated material that has been removed from its natural state or otherwise been subjected to human manipulation.
- Isolated material such as the hairpin oligonucleotides, may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state.
- Isolated material may be in native, chemical synthetic or recombinant form.
- the binding site for the target protein may be complementary to or comprise the entirety of a consensus or reference sequence thereof, or a part thereof. It is also envisaged that the binding site for the target protein may include a variant of a consensus or reference sequence thereof.
- the degree of identity of the sequence of the binding site to the consensus nucleic acid sequence or the reference nucleic acid sequence of the binding site should be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% and any range therein) and more particularly 95-100%.
- the hairpin oligonucleotide may of course comprise unrelated sequences, such as at a 5’ and/or 3’ end of the consensus or reference nucleic acid sequence, which may function to stabilize the molecule, such as described herein.
- the hairpin oligonucleotide provided herein specifically binds to the target protein or binds to the target protein with high affinity.
- the hairpin oligonucleotide binds to the target protein at an affinity that is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or any range therein, greater than the affinity of a DNA ligand (e.g., an unmodified double stranded DNA molecule) comprising, consisting of or consisting essentially of the binding site (e.g., a binding site of SEQ ID NOs: 3-32) of the hairpin oligonucleotide for the target protein.
- a DNA ligand e.g., an unmodified double stranded DNA molecule
- the binding site e.g., a binding site of SEQ ID NOs: 3-32
- the term “binds” refers to the interaction of the hairpin oligonucleotide with the target protein (e.g., a transcription factor, such as cMyc) and means that the interaction is dependent upon the presence of a particular structure (e.g., a binding site having a particular nucleic acid sequence) on the hairpin oligonucleotide that is recognised by the target protein.
- the hairpin oligonucleotide recognizes and binds to a specific target protein rather than to molecules or proteins generally.
- the term “specifically binds” shall be taken to mean that the binding interaction between a hairpin oligonucleotide disclosed herein and a target protein described herein (e.g., cMyc) is dependent on detection of the target protein by the hairpin oligonucleotide. Accordingly, the hairpin oligonucleotide preferentially binds or recognizes the target protein even when present in a mixture of other molecules, proteins, nucleic acids or organisms.
- high affinity and “relatively high affinity” are used interchangeably herein and refer to a binding affinity between a hairpin oligonucleotide and the target protein of interest with a K D of at least about 10 -6 M, more particularly at least about 10 -7 M, more particularly at least about 10 -8 M, even more particularly at least about 10 -9 M and yet even more particularly between about 10 -8 M to about 10 -10 M.
- the terms “low affinity” and “relatively low affinity” are used interchangeably herein and refer to a binding affinity between a hairpin oligonucleotide and the target protein of interest with a K D of less than about 10 -6 M, preferably less than about 10 -5 M, more preferably less than about 10 -4 M and even more preferably between about 10 -2 M to about 10- 4 M.
- the determination of such affinity may be conducted under standard competitive binding immunoassay procedures, as are known in the art, such as electrophoretic shift assay (EMSA), ELISA and surface plasmon resonance (SPR).
- the hairpin oligonucleotides of the present disclosure inhibit the binding between the target protein and one or more of its binding sites (e.g., a consensus DNA binding site, such as the DNA binding sites of SEQ ID NOs: 3-32).
- the hairpin oligonucleotide has a half maximal inhibitory concentration (IC 50 ) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for inhibiting binding of the target protein (e.g., cMyc or cMyc:Max) to a binding site thereof (e.g., 5'-CACGTG-3' or 5'-GAGCACGTGGTT-3').
- IC 50 half maximal inhibitory concentration
- the hairpin oligonucleotide has a half maximal inhibitory concentration (IC50) of between about 1 nM and about 500 nM (e.g., about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500 nM or any range therein) for inhibiting binding of the target protein (e.g., cMyc or cMyc:Max) to a binding site thereof (e.g., 5'-CACGTG-3' or 5'- GAGCACGTGGTT-3').
- IC50 half maximal inhibitory concentration
- the hairpin oligonucleotide has a half maximal inhibitory concentration (IC 50 ) of about 100 nM or less for inhibiting binding of cMyc to a binding site thereof (e.g., 5'-CACGTG-3' or 5'-GAGCACGTGGTT-3'). In certain examples, the hairpin oligonucleotide has a half maximal inhibitory concentration (IC50) of about 150 nM or less for inhibiting binding of cMyc:Max to a binding site thereof (e.g., 5'-CACGTG-3' or 5'- GAGCACGTGGTT-3').
- Hairpin oligonucleotide may have nucleobase (“base”) modifications or substitutions. Such modifications can advantageously increase the binding specificity of the stem region for the target protein and/or reduce or minimise the immunogenicity or antigenicity of the hairpin oligonucleotide.
- base nucleobase
- one or more bases e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc bases inclusive of any range therein
- one or more nucleotides e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides
- one or more nucleotides at a 5’ end of the hairpin oligonucleotide are modified.
- one or more nucleotides e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides at a 3’ end of the hairpin oligonucleotide are modified.
- all bases of the oligonucleotide described herein are modified.
- no bases of the oligonucleotide described herein are modified.
- at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the bases of the hairpin oligonucleotide are modified.
- modified bases include oligonucleotides comprising one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl.
- the hairpin oligonucleotide comprises one of the following at the 2' position: O[(CH 2 )nO]mCH 3 , O(CH 2 )nOCH 3 , O(CH 2 )nNH 2 , O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10.
- modified oligonucleotides include one or more nucleotides comprising one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties.
- the modification is selected from the group consisting of a 2'-O-methyl, 2'-O-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2- O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino, fluoroarabinonucleotide, threose nucleic acid or 2'-O--(N-methlycarbamate).
- the modified base comprises a 2'-O-methyl, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'- bridge, 4'-(CH2)2-O-2'-bridge, 2'-amino, fluoroarabinonucleotide, threose nucleic acid, 2'-O-- (N-methlycarbamate) and any combination thereof.
- the modification includes 2'-methoxy (2'-O-CH3 or 2’OMe), that is, an alkoxyalkoxy group.
- the hairpin oligonucleotide includes one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 5’ end and/or a 3’ thereof that are 2’OMe modified.
- the hairpin oligonucleotide includes at least one nucleotide at a 5’ end and a 3’ thereof that are 2’OMe modified.
- the hairpin oligonucleotide includes at least one nucleotide at a 5’ end thereof that is 2’OMe modified.
- the hairpin oligonucleotide includes at least one nucleotide at a 3’ end thereof that is 2’OMe modified.
- the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3 (also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995).
- the modification includes 2'-dimethylaminooxyethoxy, that is, a O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE), or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), that is, 2'-O-CH 2 -O-CH 2 -N(CH 3 ) 2 .
- the 2'-modification may be in the arabino (up) position or ribo (down) position.
- a 2'-arabino modification is 2'-F.
- Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide.
- Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar.
- Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, US 4,981,957, US 5,118,800, US 5,319,080, US 5,359,044, US 5,393,878, US 5,446,137, US 5,466,786, US 5,514,785, US 5,519,134, US 5,567,811, US 5,576,427, US 5,591,722, US 5,597,909, US 5,610,300, US 5,627,053, US 5,639,873, US 5,646,265, US 5,658,873, US 5,670,633, US 5,792,747, and US 5,700,920.
- a further modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'- hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.
- LNAs Locked Nucleic Acids
- the linkage is a methylene (-CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2.
- LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
- Modified nucleobases include other synthetic and natural nucleobases such as, for example, 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-CC-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
- nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H- pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), and phenothiazine cytidine (1H-pyrimido[5,4- b][1,4]benzothiazin-2(3H)-one), G-clamps such as, for example, a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
- Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone.
- Further nucleobases include those disclosed in US 3,687,808, those disclosed in J.I. Kroschwitz (editor), The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, John Wiley and Sons (1990), those disclosed by Englisch et al. (1991), and those disclosed by Y.S. Sanghvi, Chapter 15: Antisense Research and Applications, pages 289-302, S.T. Crooke, B. Lebleu (editors), CRC Press, 1993.
- nucleobases are particularly useful for increasing the binding affinity of the hairpin oligonucleotide.
- These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine.
- 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 o C.
- these nucleobase substitutions are combined with 2'-O-methoxyethyl sugar modifications.
- Oligonucleotides of the present disclosure include those having modified backbones or non- natural internucleotide linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Such modifications, inclusive of a phosphorothioate backbone, may advantageously protect the hairpin oligonucleotide from digestion by nucleases and/or increase the affinity of the hairpin oligonucleotide to the target protein.
- Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is
- Oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage, that is, a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof).
- Various salts, mixed salts and free acid forms are also included.
- Modified oligonucleotide backbones that do not include a phosphorus atom therein include, for example, backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
- morpholino linkages formed in part from the sugar portion of a nucleoside
- siloxane backbones sulfide, sulfoxide and sulfone backbones
- formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
- riboacetyl backbones alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
- the hairpin oligonucleotide described herein at least partly comprises a modified backbone.
- modified backbones useful for the invention can include those which comprise a phosphorothioate, a non-bridging oxygen atom substituting a sulfur atom, a phosphonate such as a methylphosphonate, a phosphodiester, a phosphoromorpholidate, a phosphoropiperazidate, amides, methylene(methylamino), fromacetal, thioformacetal, a peptide nucleic acid or a phosphoroamidate such as a morpholino phosphorodiamidate (PMO), N3’-P5’ phosphoramidite or thiophosphoroamidite.
- PMO morpholino phosphorodiamidate
- the oligonucleotide comprises a 5’ region comprising one or more bases which are modified and/or which have a modified backbone and a 3’ region comprising one or more bases which are modified and/or which have a modified backbone.
- all internucleotide linkages of the hairpin oligonucleotide described herein are modified or comprise a modification, such as a phosphorothioate modification.
- no internucleotide linkages of the hairpin oligonucleotide described herein are modified or comprise a modification.
- the hairpin oligonucleotide comprises one or a plurality of phosphorothioate internucleotide linkages.
- all internucleotide linkages of the hairpin oligonucleotide comprise a phosphorothioate modification.
- at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the internucleotide linkages of the hairpin oligonucleotide are modified or non-natural.
- the oligonucleotide has/is a ribonucleic acid, deoxyribonucleic acid, a DNA phosphorothioate, an RNA phosphorothioate, 2'-O-methyl- oligonucleotide, 2'-O-methyl-oligodeoxyribonucleotide, 2'-O-hydrocarbyl ribonucleic acid, 2'- O-hydrocarbyl DNA, 2'-O-hydrocarbyl RNA phosphorothioate, 2'-O-hydrocarbyl DNA phosphorothioate, 2'-F-phosphorothioate, 2'-F-phosphodiester, 2'-methoxyethyl phosphorothioate, 2-methoxyethyl phosphodiester, deoxy methylene(methylimino) (deoxy MMI), 2'-O-hydrocarby MMI, deoxy-methylphos-
- the hairpin oligonucleotides described herein can be utilised to bind the target protein, such as a transcription factor, in cells and thereby inhibit a biological activity thereof.
- the hairpin oligonucleotides may be administered to a subject in need thereof to prevent a disease, disorder or condition associated with or dependent on increased activity and/or expression of the target protein. Therefore, in one form, the present disclosure provides a method of inhibiting a target protein of a cell, said method including the step of contacting the cell with an effective amount the hairpin oligonucleotide described herein.
- the present disclosure provides for the use of the hairpin oligonucleotide or the pharmaceutical composition provided herein in the manufacture of a medicament for inhibiting a target protein of a cell.
- the present disclosure provides a hairpin oligonucleotide or a pharmaceutical composition described herein for use in inhibiting a target protein of a cell. It is contemplated that such methods may be performed in relation to cells in vitro, in vivo and/or ex vivo.
- the method is performed in vitro, such as with cancer cells or patient-derived xenografts isolated from a subject, an organoid or a stem cell-derived (e.g., induced pluripotent stem cell(iPSC)-derived) cell, as described herein.
- the present method includes the earlier step of isolating the cells expressing the target protein from a subject.
- the present method is performed in vivo in a subject.
- the term “effective amount” and the like refers to an amount of the hairpin oligonucleotide or the pharmaceutical composition that is sufficient to induce a desired physiological outcome (e.g., at least partly inhibiting a target protein’s activity).
- an effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period which the individual dosage unit is to be used, the bioavailability of the composition, the route of administration, etc.
- the present disclosure provides a method of preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject, said method including the step of administering to the subject a therapeutically effective amount of the hairpin oligonucleotide or the pharmaceutical composition described herein to thereby prevent, ameliorate or treat the disease, disorder or condition.
- the present disclosure also provides for the use of the hairpin oligonucleotide or the pharmaceutical composition described herein in the manufacture of a medicament for preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject.
- the present disclosure also provides for a hairpin oligonucleotide or a pharmaceutical composition described herein for use in preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject.
- the term “subject”, “patient” and “individual” includes, but is not limited to, mammals, inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs).
- the subject is a human.
- the subject is a female human.
- the subject is a male human.
- “treating”, “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a disease, disorder or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject.
- preventing refers to a course of action initiated before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject.
- a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.
- hairpin oligonucleotide refers to any improvement in the disease state of a patient suffering from the disease, disorder or condition described herein by administering to a subject in need thereof a hairpin oligonucleotide according to the present disclosure. Such an improvement may also be seen as a slowing or stopping of the progression of the disease, disorder or condition in the subject.
- Hairpin oligonucleotides of the present disclosure can be used to target any protein of interest that binds or interacts with a particular nucleic acid sequence, inclusive of secondary, tertiary and quaternary structures formed thereby.
- the target protein may be extracellularly expressed or may be intracellularly expressed.
- the hairpin oligonucleotide is used to modify a trait of an animal, more typically to treat or prevent a disease, disorder or condition.
- the disease, disorder or condition will benefit from inhibition of the target protein following administration of the hairpin oligonucleotide.
- the disease, disorder or condition may be at least partly caused by an increased expression and/or activity of the target protein (e.g., increased expression and/or activity of cMyc).
- cancers for example breast cancer, ovarian cancer, cancers of the central nervous system, gastrointestinal cancer, bladder cancer, skin cancer, lung cancer, head and neck cancers, haematological and lymphoid cancers, bone cancer
- rare genetic diseases for example, neuromuscular and neurological diseases (for example, spinal muscular atrophy, Amyotrophic Lateral Sclerosis, Duchenne muscular dystrophy, Huntington’s disease, Batten disease, Parkinson’s disease, amyotrophic lateral sclerosis, Ataxia-telangiectasia, cerebral palsy) viruses (for example, cytomegalovirus, hepatitis C virus, Ebola haemorrhagic fever virus, human immunodeficiency virus, coronaviruses), cardiovascular disease (for example, familial hypercholesterolemia, hypertriglyceridemia), autoimmune and inflammatory diseases (for example arthritis, lupus
- the disease, disorder or condition is a cancer.
- Cancers may include any aggressive or potentially aggressive cancers, tumours or other malignancies such as listed in the NCI Cancer Index at http://www.cancer.gov/types, including all major cancer forms such as sarcomas, carcinomas, lymphomas, leukaemias and blastomas, although without limitation thereto.
- These may include solid cancers and haematological cancers, and more particularly breast cancer, lung cancer inclusive of lung adenocarcinoma, cancers of the reproductive system inclusive of ovarian cancer, cervical cancer, uterine cancer and prostate cancer, cancers of the brain and nervous system, head and neck cancers, gastrointestinal cancers inclusive of colon cancer, colorectal cancer and gastric cancer, liver cancer, kidney cancer, skin cancers such as melanoma and skin carcinomas, blood cell cancers inclusive of lymphoid cancers and myelomonocytic cancers, cancers of the endocrine system such as pancreatic cancer and pituitary cancers, musculoskeletal cancers inclusive of bone and soft tissue cancers, although without limitation thereto.
- the cancer can be a breast cancer, which may include any aggressive breast cancers and cancer subtypes known in the art, such as triple negative breast cancer, grade 2 breast cancer, grade 3 breast cancer, lymph node positive (LN+) breast cancer, HER2 positive (HER2+) breast cancer, PR negative (PR-) breast cancer, PR positive (PR + ) breast cancer, ER negative (ER-) breast cancer and ER positive (ER+) breast cancer.
- the breast cancer is or comprises triple negative breast cancer.
- the term “therapeutically effective amount” describes a quantity of a specified agent, such as a hairpin oligonucleotide or a composition described herein, sufficient to achieve a desired effect in a subject being treated with that agent or composition.
- this can be the amount of a hairpin oligonucleotide and optionally one or more further therapeutic agents, necessary to reduce, alleviate and/or prevent a disease, disorder or condition associated with the target protein in question.
- a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of such a disease, disorder or condition. More particularly, a “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression, such as progressive vision loss and blindness.
- a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject.
- an agent useful for reducing, alleviating and/or preventing the diseases, disorders and conditions described herein will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition (e.g., disease progression), and the manner of administration of the therapeutic composition.
- the methods described herein further include the step of administering a further therapeutic agent to the subject (i.e., in addition to the hairpin oligonucleotide).
- the hairpin oligonucleotide may be administered alone (i.e., monotherapy) or alternatively be administered in combination with the further therapeutic agent (e.g., a further anti-cancer agent) which aims to treat or prevent a disease, disorder or condition described herein.
- the hairpin oligonucleotide described herein may be co-administered with (simultaneously or sequentially) a further treatment of a cancer (e.g., an anti-cancer agent).
- a cancer e.g., an anti-cancer agent
- treatments include drug therapy, chemotherapy, antibody, nucleic acid and other biomolecular therapies, radiation therapy, surgery, nutritional therapy, relaxation or meditational therapy and other natural or holistic therapies, although without limitation thereto.
- drugs, biomolecules e.g., antibodies, inhibitory nucleic acids such as siRNA
- chemotherapeutic agents are referred to herein as “anti-cancer therapeutic agents” or “anti-cancer agents”.
- the treatment is or comprises one or more of chemotherapy, radiation therapy, molecularly targeted therapy and immunotherapy.
- the present methods further include the step of administering a therapeutically effective amount of an anti-cancer agent to the subject.
- the methods of the present disclosure may further include the earlier or initial step of identifying the presence of the disease, disorder or condition associated with the target protein in the subject.
- the methods described herein may include the earlier or initial step of identifying a cancer or a cancer cell as a target protein-dependent cancer or cancer cell.
- the methods of this disclosure may include the initial step of determining an activity level and/or an expression level of the target protein of a cancer cell or the subject’s cancer, such as from a sample (e.g., a biopsy sample or a biological sample) obtained from the subject or the subject’s cancer.
- a determining step may be performed by any means or method of testing known in the art.
- Target proteins As used herein, a “target”, such as a “target protein” or “target polypeptide”, refers to a molecule upon which a hairpin oligonucleotide of the invention directly or indirectly exerts its effects (e.g., specifically binds or contacts).
- the hairpin oligonucleotide of the present disclosure or a portion thereof and the target protein, or a portion thereof, are able to bind under physiological conditions.
- protein is meant an amino acid polymer.
- the amino acids may be natural or non-natural amino acids, D- or L-amino acids, as are well understood in the art.
- a “peptide” is generally referred to as a protein typically having no more than fifty (50) amino acids and a polypeptide is generally referred to as a protein typically having more than fifty (50) amino acids.
- the target protein may be a nucleic acid binding protein that can specifically bind to a certain nucleic acid sequence.
- the target protein is a DNA- binding protein.
- the target protein is an RNA-binding protein.
- the target protein is capable of binding both DNA and RNA.
- Conventional methods for detecting nucleic acid binding proteins, such as transcription factors, include electrophoretic shift assay (EMSA), supershift EMSA, and ELISA-based techniques.
- a nucleic acid binding protein may be a complex of two or more individual molecules (e.g., cMyc:Max). Such complexes are commonly referred to as “homodimers”, “heterodimers”, “homotype complexes” and “heterotype complexes”. Such a complex suitably comprises a number of individual components joined together by covalent bonds or non-covalent interactions.
- the target protein may be a transcription factor that is capable of binding or specifically binding, such as with high affinity, to the binding site of the hairpin oligonucleotide.
- the hairpin oligonucleotide can inhibit transcription factor function, for example, by interfering with binding thereof to DNA.
- transcription factor as used herein means a protein that possesses a biological function that includes regulation, such as initiation or inhibition/repression, of the transcription of one or more genes. That is, a transcription factor is a protein that possesses a DNA-binding domain (DBD) that allows the protein to bind a specific sequence of DNA (e.g., an enhancer element or promoter sequence).
- DBD DNA-binding domain
- the transcription factor's presence can aid in initiation of transcription by stabilizing transcription initiation complex formation and/or activity, for example.
- Transcription factors can also inhibit transcription by blocking (i.e., a repressor) the recruitment of RNA polymerase to one or more specific genes, such as by binding to a silencer sequence.
- Transcription factors also bind to regulatory DNA sequences, such as enhancer sequences, that can be many hundreds of base pairs downstream or upstream from the transcribed gene. Transcription factors can perform the transcription controlling function either alone or in combination with other proteins, such as by forming an activation complex, and can aid in recruiting RNA polymerase and related proteins to the transcription initiation start site.
- the binding site of the hairpin oligonucleotide may be or comprise a regulatory DNA sequence, such as a promoter sequence, a silencer sequence or an enhancer sequence, or a fragment or variant thereof.
- promoter sequence refers to a polynucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3’-direction) coding sequence.
- the term “enhancer sequence” refers to a sequence capable of increasing gene expression. Such sequences may be located upstream, intronically or downstream of the region to be transcribed.
- Enhancement of gene expression by enhancer sequences can be achieved through a variety of mechanisms including, but not limited to, increased transcription efficiency, stabilization of mature mRNA, and translation enhancement.
- the term “silencer sequence” refers to a polynucleotide region comprising a DNA regulatory sequence comprising one or more sequence-specific binding sites for a repressive transcription factor or repressor that can suppress or inhibit transcription from an otherwise active promoter.
- the binding site of the hairpin oligonucleotide may include one or more additional nucleotides (e.g., 1, 2, 3, 4, 5, etc nucleotides) either side (i.e., 5’ or 3’ end) thereof that are naturally or normally found in a genomic sequence that includes said binding site.
- flanking nucleotide sequences may assist in improving the affinity of the target protein for the binding site. It is further contemplated that the loop region may at least in part facilitate binding of the target protein to the hairpin oligonucleotide.
- the loop region may include a portion, such as a 5’ end or a 3’ end, of the binding site in question.
- the loop region together with the stem region forms a secondary structure that is capable of binding or interacting with a target protein
- Transcription factors include a wide number of proteins, excluding RNA polymerase, that initiate and regulate the transcription of genes.
- Exemplary transcription factors include, but are not limited to, AAF, ab1, ADA2, ADA-NF1, AF-1, AFP1, AhR, AIIN3, ALL-1, alpha-CBF, alpha-CP1, alpha-CP2a, alpha-CP2b, alphaHo, alphaH2-alphaH3, Alx-4, aMEF-2, AML1, AML1a, AML1b, AML1c, AML1DeltaN, AML2, AML3, AML3a, AML3b, AMY-1 L, A-Myb, ANF, AP-1, AP-2alphaA, AP-2alphaB, AP- 2beta, AP-2gamma, AP-3 (1), AP-3 (2), AP-4, AP-5, APC, AR, AREB6, Arnt, Arnt (774 M form), ARP-1, ATBF1-A, ATBF1-B, ATF, ATF-1, ATF-2, ATF-3, ATF-3deltaZIP
- ENKTF-1 prot., ENKTF-1, EPAS1, epsilonF1, ER, Erg-1, Erg- 2, ERR1, ERR2, ETF, Ets-1, Ets-1 delta Vil, Ets-2, Evx-1, F2F, factor 2, Factor name, FBP, f- EBP, FKBP59, FKHL18, FKHRL1P2, Fli-1, Fos, FOXB1, FOXC1, FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXE1, FOXE3, FOXF1, FOXF2, FOXG1a, FOXG1b, FOXG1c, FOXH1, FOXI1, FOXJ1a, FOXJ1b, FOXJ2 (long isoform), FOXJ2 (short isoform), FOXJ3, FOXK1a, FOXK1b, FOXK1c, FOXL1, FOXM1a, FOXM1b, FOXM1c,
- the transcription factor can be an E-box transcription factor capable of binding an E-box transcription factor binding site or domain (i.e., an enhancer box or E-box).
- an E-box is a DNA response element found in some eukaryotes that acts as a protein binding site and has been shown to regulate gene expression in a range of cells and tissues.
- the binding site of the hairpin oligonucleotide may comprise, consist of or consist essentially of an E-box binding site, as are known in the art.
- binding sites comprise, consist of or consist essentially of a nucleotide sequence of 5'-CANNTG-3' (SEQ ID NO: 4; i.e., a consensus E-box binding site sequence), wherein N can be any nucleotide (e.g., A, C, T, G or U).
- the E-box binding site comprises, consists of or consists essentially of a nucleotide sequence of 5'-CAC[GA]TG-3' (SEQ ID NO: 5) (wherein [GA] or [G/A] indicates a nucleotide variation of G or A at this position) or more particularly 5'- CACGTG-3' (SEQ ID NO: 6) or 5'-CACATG-3' (SEQ ID NO: 7), or a nucleotide sequence having one or two substitutions, deletions, or insertions therein.
- substitutions, deletions, or insertions may be any substitution, deletion, or insertion of a single nucleotide such that the E- box transcription factor binding site retains at least one of its endogenous functions (e.g., an ability to bind an E-box transcription factor).
- one or more of the thymine residues may be replaced or substituted by uracil residues or vice versa (e.g., 5'-CANNUG-3' (SEQ ID NO: 14), 5'-CAC[GA]UG-3' (SEQ ID NO: 11), 5'-CACGUG-3' (SEQ ID NO: 12) or 5'-CACAUG-3' (SEQ ID NO: 13)).
- the E-box binding site comprises, consists of, consists essentially of a nucleotide sequence of 5'- CACGTT-3' (SEQ ID NO: 8), 5'- CAGCTT-3' (SEQ ID NO: 9) or 5'-CACCTCGTGAC-3' (SEQ ID NO: 10), or a nucleotide sequence having one or two substitutions, deletions, or insertions therein.
- one or more of the thymine residues may be replaced or substituted by uracil residues or vice versa (e.g., 5'- CACGUU-3' (SEQ ID NO: 15), 5'- CACGUT-3' (SEQ ID NO: 16), 5'- CACGTU-3' (SEQ ID NO: 17), 5'- CAGCUU-3' (SEQ ID NO: 18), 5'- CAGCUT-3' (SEQ ID NO: 19), 5'- CAGCTU-3' (SEQ ID NO: 20), 5'-CACCUCGUGAC-3' (SEQ ID NO: 21), 5'-CACCTCGUGAC-3' (SEQ ID NO: 22) or 5'-CACCUCGTGAC-3' (SEQ ID NO: 23)).
- 5'- CACGUU-3' SEQ ID NO: 15
- 5'- CACGUT-3' SEQ ID NO: 16
- 5'- CACGTU-3' SEQ ID NO: 17
- 5'- CAGCUU-3' SEQ ID
- Exemplary E-box transcription factors include Myc/Max, Fos/Jun, HIF1 ⁇ / ⁇ , MITF, MyoD, HES family, Hey family, ID1/2/3, E2 family, Twist, AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATF1, ATF2, ATF4, ATF5, ATF6, ATF7, ATOH1, ATOH7, ATOH8, BACH1, BACH2, BATF, BATF2, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, CREB1, CREB3, CREB3L1, CREB3L2, CREB3L3, CREB3L4, CREB5, CREBL1, CREM, E4BP4, EPAS1, FERD3L, FIGLA, FOSL1, FOSL2, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, H
- the target protein is cMyc (inclusive of the cMyc-Max protein complex).
- cMyc is a protooncogene, which is overexpressed in a wide range of human cancers. When it is specifically mutated, or overexpressed, it increases cell proliferation and functions as an oncogene.
- the MYC gene encodes for a transcription factor that regulates expression of 15% of all genes through binding on Enhancer Box sequences (E-boxes) and recruiting histone acetyltransferases (HATs).
- E-boxes Enhancer Box sequences
- HATs histone acetyltransferases
- Myc-family transcription factors contain the bHLH/LZ (basic Helix- Loop-Helix Leucine Zipper) domain.
- cMyc protein or simply cMyc relates to human cMyc.
- accession number P01106 UniProtKB.
- cMyc protein (SEQ ID NO:2) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQSELQPPAPSEDIWKKFELLPTPP LSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPD DETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQD LSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVL HEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRC HVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHN VLERQRRNELKRSFFALRD
- the binding site of the hairpin oligonucleotide may comprise, consist of or consist essentially of the nucleic acid sequence of 5'-CAC[GA]TG-3' (SEQ ID NO: 5) or 5'-CAC[GA]UG-3' (SEQ ID NO: 11).
- the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of a nucleic acid sequence of 5'-CACGTG-3' (SEQ ID NO: 6), 5'- CACGUG-3' (SEQ ID NO: 12), 5'-CACATG-3' (SEQ ID NO: 7), 5'-CACAUG-3' (SEQ ID NO: 13) or a fragment, variant or derivative thereof.
- the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-CACGTG-3' (SEQ ID NO: 6) or 5'-CACGUG-3' (SEQ ID NO: 12).
- the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-CACATG-3' (SEQ ID NO: 7) or 5'-CACAUG-3' (SEQ ID NO: 13).
- the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-GAGCAC[GA]UGGUU-3' (SEQ ID NO: 31), wherein one or more of the U nucleotides may be a T.
- the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-GAGCACGUGGUU-3' (SEQ ID NO: 3) or a fragment, variant or derivative thereof.
- Such variants may include the nucleotide sequence of 5'-GAGCACAUGGUU-3' (SEQ ID NO: 32), wherein one or more of the U nucleotides may be a T.
- the nucleotide sequence of SEQ ID NO: 3 represents the NPM1 gene’s E-box promoter sequence inclusive of adjacent, flanking or surrounding base pairs, which is regarded as a high affinity binding site for cMyc.
- one or more of the U nucleotides may be a T (e.g., 5'-GAGCACGTGGUU-3' (SEQ ID NO: 24), 5'- GAGCACGTGGTU-3' (SEQ ID NO: 25), 5'-GAGCACGTGGTT-3' (SEQ ID NO: 26), 5'- GAGCACGTGGUT-3' (SEQ ID NO: 27), 5'-GAGCACGUGGTU-3' (SEQ ID NO: 28), 5'- GAGCACGUGGUT-3' (SEQ ID NO: 29) or 5'-GAGCACGUGGTT-3' (SEQ ID NO: 30)).
- T e.g., 5'-GAGCACGTGGUU-3' (SEQ ID NO: 24), 5'- GAGCACGTGGTU-3' (SEQ ID NO: 25), 5'-GAGCACGTGGTT-3' (SEQ ID NO: 26), 5'- GAGCACGTGGUT-3' (SEQ ID NO: 27), 5'-GAGCACGUGGTU-3
- the hairpin oligonucleotide in a single stranded form, comprises, consists of or consists essentially of the nucleic acid sequence of 5'- GAGCACGUGGUUAAAAAACCACGUGCUC-3' (SEQ ID NO: 1; Figure 1) or a fragment, variant or derivative thereof.
- SEQ ID NO: 1 the nucleic acid sequence of 5'- GAGCACGUGGUUAAAAAACCACGUGCUC-3'
- SEQ ID NO: 1 may be a T.
- one or more of the nucleotides and/or the internucleotide linkages of SEQ ID NO: 1 can be modified, such as described herein.
- all of the internucleotide linkages of SEQ ID NO: 1 comprise a phosphorothioate modification.
- the 3’ terminal cytosine (C) residue of SEQ ID NO: 1 comprises a 2’OMe modification.
- the 5’ terminal guanine (G) residue of SEQ ID NO: 1 comprises a 2’OMe modification.
- the 3’ terminal cytosine (C) residue and the 5’ terminal guanine (G) residue of SEQ ID NO: 1 comprises a 2’OMe modification.
- all of the internucleotide linkages of SEQ ID NO: 1 comprise a phosphorothioate modification and all of the nucleic acid bases of SEQ ID NO: 1 are modified, such as by way of a 2’OMe modification (e.g., 5′- mG*mA*mG*mC*mA*mC*mG*mU*mG*mG*mU*mU*mA*mA*mA*mA*mA*mA*mA*mC *mC*mA*mC*mG*mU*mG*mC*mU*mC-3′; phosphorothioate modified internucleotide linkages are indicated by and 2′-OMe modified nucleic acid bases are indicated by “m”).
- a 2’OMe modification e.g., 5′- mG*mA*mG*mC*mA*mC*mG*mU*mG*mG*mU*mU*mA*
- cMyc may function in conjunction with Max. These two transcription factors can form a heterodimer on the promoter of a target gene. There are low and high affinity targets for cMyc:Max and thus cMyc’s expression is typically fine-tuned to only drive activation of specific promoters in the context of cell type.
- the hairpin oligonucleotide can inhibit Myc/Max activity or function, such as by interfering with Myc/Max binding to DNA (e.g., a E-box transcription factor binding site or another DNA binding site, such as those described herein).
- the phrase “inhibits Myc/Max activity” or variations thereof means that after administration of an hairpin oligonucleotide described herein, such as that of SEQ ID NO: 1, to an animal or a cell, an activity of Myc/Max in the animal or cell, such as the ability of this protein complex to bind E boxes, is abrogated or reduced.
- the activity of Myc/Max is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of the activity in the absence of the hairpin oligonucleotide.
- the pharmaceutical compositions described herein comprise an acceptable carrier, diluent or excipient.
- acceptable carrier diluent or excipient
- diluent or excipient a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration.
- a variety of carriers, diluent and excipients well known in the art may be used.
- These may be selected from a group including sugars, starches, cellulose and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water.
- a useful reference describing acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference.
- Hairpin oligonucleotides of the disclosure may be admixed, encapsulated, conjugated (such as fused) or otherwise associated with other molecules, molecule structures or mixtures of compounds, resulting in, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption.
- the hairpin oligonucleotides may be formulated as pharmaceutically acceptable salts, esters, or salts of the esters, or any other compounds which, upon administration are capable of providing (directly or indirectly) the biologically active metabolite.
- pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the oligonucleotide that retain the desired biological activities of the parent compounds and do not impart undesired toxicological effects upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860. It is further envisaged that the hairpin oligonucleotides provided herein may be prodrugs or pharmaceutically acceptable salts of the prodrugs, or other bioequivalents.
- prodrugs refers to therapeutic agents that are prepared in an inactive form that is converted to an active form (i.e., a drug) upon administration by the action of endogenous enzymes or other chemicals and/or conditions.
- prodrug forms of the oligonucleotide of the disclosure are prepared as SATE [(S acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510, WO 94/26764 and US 5,770,713.
- a prodrug may, for example, be converted within the body, such as by hydrolysis in the blood, into its active form that has medical effects.
- Pharmaceutical acceptable prodrugs are described in T. Higuchi and V.
- the hairpin oligonucleotides of the present disclosure can be complexed with a complexing agent to increase cellular uptake thereof.
- a complexing agent includes cationic lipids.
- Cationic lipids can be used to deliver oligonucleotides to cells.
- the term “cationic lipid” includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells.
- cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof.
- Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms. Exemplary straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
- counterions anions
- cationic lipids examples include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINE TM (e.g., LIPOFECTAMINE TM 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).
- PAMAM polyamidoamine
- DOPE Lipofectase
- LIPOFECTAMINE TM e.g., LIPOFECTAMINE TM 2000
- DOPE Cytofectin
- Cytofectin Gilead Sciences, Foster City, Calif.
- Eufectins JBL, San Luis Obispo, Calif.
- Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3.beta.-[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB).
- DOTMA N-[
- Oligonucleotides can also be complexed with, e.g., poly (L-lysine) or avidin and lipids may, or may not, be included in this mixture, e.g., steryl-poly (L-lysine).
- Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., US 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al., 1996; Hope et al., 1998).
- lipid compositions are also known in the art and include, for example, those taught in US 4,235,871; US 4,501,728; 4,837,028; 4,737,323.
- lipid compositions can further comprise agents (e.g., viral proteins) to enhance lipid-mediated transfections of oligonucleotides.
- agents e.g., viral proteins
- N-substituted glycine oligonucleotides peptoids
- a composition for delivering hairpin oligonucleotides of the present disclosure comprises a peptide having from between about one to about four basic residues.
- amino acids with basic side chains e.g., lysine, arginine, histidine
- acidic side chains e.g., aspartic acid, glutamic acid
- uncharged polar side chains e.g., glycine (can also be considered non-polar
- asparagine, glutamine, serine, threonine, tyrosine, cysteine nonpolar side chains
- nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
- beta-branched side chains e.g., threonine, valine, isoleucine
- aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
- the hairpin oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide”.
- the composition includes an oligonucleotide and a covalently attached transporting peptide.
- the hairpin oligonucleotide is attached to a targeting moiety such as N- acetylgalactosamine (GalNAc), an antibody, an antibody-like molecule or aptamer (see, for example, Toloue and Ford (2011) and Esposito et al. (2016)).
- a targeting moiety such as N- acetylgalactosamine (GalNAc), an antibody, an antibody-like molecule or aptamer (see, for example, Toloue and Ford (2011) and Esposito et al. (2016)).
- GalNAc N- acetylgalactosamine
- Administration Any safe route of administration may be employed, including oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intranasal, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal and transdermal administration, although without limitation thereto
- the oligonucleotide of the disclosure is administered systemically.
- systemic administration is a route of administration that is either enteral or parenteral.
- Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like.
- These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose.
- compositions may be presented as discrete units such as capsules, sachets, functional foods/feeds or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
- Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients.
- compositions are prepared by uniformly and intimately admixing the agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
- the above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as effective.
- the dose administered to a subject should be sufficient to effect a beneficial response in a subject over an appropriate period of time.
- the quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner.
- the present disclosure provides a method for selecting a hairpin oligonucleotide for inhibiting a target protein, said method including the steps of: (a) producing one or more candidate hairpin oligonucleotides comprising a loop region and a double-stranded linear region, wherein the double-stranded linear region comprises a binding or recognition site for the target protein; (b) testing the ability of the one or more candidate hairpin oligonucleotides to bind and/or inhibit the target protein, and (c) selecting a hairpin oligonucleotide which binds and/or inhibits the target protein.
- the hairpin oligonucleotide, inclusive of modifications thereof, and the target protein are that hereinbefore described.
- factors to be considered when producing a hairpin oligonucleotide depend on the purpose of the oligonucleotide but include features such as strength and stability of the oligonucleotide- target protein interaction, such as the secondary structure of the hairpin oligonucleotide, thermodynamic stability, the position of the DNA-binding domain of the target protein, and/or functional motifs.
- the phrase “inhibits the target protein” or “inhibits an activity of the target protein” or variations thereof means that after administration of a hairpin oligonucleotide to an animal or cell, the animal or cell is not able to elicit a target protein-based biological or cellular response or is only able to elicit a reduced or partial target protein-based biological or cellular response, such as those biological or cellular responses that are dependent upon the target protein binding to endogenous nucleic acid, when compared to a control or reference sample (e.g., control cells not treated with the candidate oligonucleotide).
- a control or reference sample e.g., control cells not treated with the candidate oligonucleotide
- the present method may include a further step that measures or detects a change in one or more biological activities of the target protein in a cell or an animal in response to the candidate oligonucleotide(s).
- the activity of the target protein is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of the activity in the absence of the candidate oligonucleotide (e.g., untreated control cells).
- the level of activity of a target protein may also be compared to a reference or threshold level thereof.
- any of the methods disclosed herein may comprise a step of establishing a reference or threshold level activity of the target protein.
- candidate hairpin oligonucleotides can be tested for their desired activity using standard procedures in the art. This may involve administering the candidate to cells in vitro expressing the target protein and measuring binding and/or inhibition thereof by the candidate hairpin oligonucleotide (e.g., analysing the amount of transcription of a target gene of the target protein, such as RNA and/or protein) and/or utilising one or more functional assays (e.g., cell growth and proliferation, apoptosis, cellular differentiation) to determine whether the activity thereof changes after exposure to a candidate hairpin oligonucleotide.
- the candidate oligonucleotide is administered to an animal, and the animal screened for the amount of target protein inhibition.
- the candidate hairpin oligonucleotide is tested for its ability to bind or hybridize to a target protein, such as by those methods described herein (e.g., assess affinity by EMSA assay).
- inhibitory activity of the candidate hairpin oligonucleotides, and more particularly those containing a binding site for a transcription factor can be assessed by mRNA reverse transcription quantitative real-time PCR (RT-qPCR) to determine expression of a target gene.
- RT-qPCR mRNA reverse transcription quantitative real-time PCR
- RNA can be extracted and purified from cells which have been incubated with a candidate oligonucleotide.
- cDNA is then synthesized from isolated RNA and RT-qPCR can be performed, using methods and reagents known the art.
- Protein products and/or downstream cell signalling (and/or one or more markers thereof) of a target gene may alternatively or additionally be determined, such as by any means known in the art, including, but not limited to, ELISA, western blot, mass spectrometry, proteomics, immunoprecipitation and immunostaining.
- the candidate hairpin oligonucleotide possesses or displays little or no significant off- target and/or nonspecific effects. It is also contemplated that the candidate hairpin oligonucleotide may be rationally designed or engineered de novo based on desired or predicted structural characteristics or features that indicate the candidate oligonucleotide could bind and/or block or inhibit one or more biological activities of the target protein.
- An initial step of the method may include identifying a plurality of candidate oligonucleotides that are selected according to broad structural and/or functional attributes, such as an ability to bind or specifically bind the target protein. Additionally, the present method may further include one or more of the steps of: selecting the candidate hairpin oligonucleotide that binds (e.g., specifically binds with high affinity) and/or modulates the expression and/or the activity of the target protein; isolating or purifying the candidate hairpin oligonucleotide; formulating the candidate hairpin oligonucleotide into a pharmaceutical formulation; and adding the candidate hairpin oligonucleotide or the pharmaceutical formulation to packaging and/or a container.
- selecting the candidate hairpin oligonucleotide that binds e.g., specifically binds with high affinity
- modulates the expression and/or the activity of the target protein isolating or purifying the candidate hairpin oligonucleotide
- the present disclosure provides a hairpin oligonucleotide designed, selected or identified by the present methods. So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples.
- Examples Example 1. This Example tested the ability of the DRpinMyc hairpin oligonucleotide to bind and functionally inhibit the cMyc/Max protein complex.
- the Drpin oligonucleotide design described herein differs from antisense oligonucleotides as it is designed to bind proteins and not DNA or RNA. Drpin is a single stranded, self folding, synthetic RNA oligonucleotide, as described in more detail above.
- the oligonucleotide folds into a hairpin, which usually has a fully Watson and Crick base pairing stem or contains nucleotides that will induce a specific secondary structure, which will be recognised by a target protein (see Figure 1).
- cMyc was chosen as the target protein to validate this technology with.
- the Myc family in humans is composed c-myc (MYC), l-myc (MYCL), and n-myc (MYCN).
- C-MYC is the best characterised as it is involved in up to 70% of human cancers. N-MYC has been shown to be involved in brain cancer, prostate cancers and well a blood cancers.
- cMyc and c-Myc:Max was incubated with 10 nM of labelled oligo for15 min at 37° C in a buffer consisting of 10 mM Tris-HCI (pH8.0), 100 mM NaCl, 0.01 % IGEPAL, 125 mM EDTA and 100 ng/ ⁇ L BSA. Samples were separated by electrophoresis on a 10% PAGE gel in TBE buffer for 60 min at 80V at 4° C. Molecular dynamics and in silico modelling Drpin Modelling The Drpin oligonucleotide secondary structure was predicted by MC-Fold, accessible as a web server at https://www.major.iric.ca/MC-Fold/.
- RNA tertiary structure models of Drpin were generated by providing the sequence and MC-Fold secondary structure to RNAcomposer at https://rnacomposer.cs.put.poznan.pl/. Conversion of the RNAComposer tertiary structure to the R P and S p Phosphorothioate with 2′-O-Methyl chemistry was performed with PyMOL from Schrödinger, Inc (Schrödinger, L. & DeLano, W., 2020. PyMOL, Available at: http://www.pymol.org/pymol.). Molecular Dynamics Setup and Simulation A novel force field library was required for the ribonucleotide chemistry used in Drpin.
- the library was created using the R.E.D. server at https://upjv.q4md-forcefieldtools.org/.
- the Drpin model was solvated in 0.15 M NaCl with TIP3P water molecules added to a padding distance of 15 ⁇ in a truncated octahedron in tleap from the Ambertools16 suite.
- Atomistic Molecular Dynamics simulations were performed at 300 K for 1 ⁇ s for RNA and both stereoisomers of Drpin using pmemd.cuda from the Amber 2020 package. Trajectory Analysis Cpptraj from AmberTools 16 was used to analyse simulation trajectories.
- K-means Clustering was calculated by sorting into two clusters based on intra-cluster RMSD measured via Davies- Bouldin Index and inter-cluster RMSD measured via pseudo-F. 3D structures of oligomers were processed, and figures were generated with ChimeraX from the University of California San Francisco. Western blotting Western/Immuno blots were performed as described previously (Bolderson et al., 2014, Nucleic Acids Res., 42, 6326–6336) and visualized using an Odyssey infrared imaging system. When necessary, immunoblots were quantified using ImageJ software and normalized to actin.
- DrpinMyc Transfections of Drpin were performed using Lipofectamine RNAimax (Life Technologies) as per manufacturer’s instructions for siRNA transfection.
- Cell culture Cells were maintained in Roswell Park Memorial Institute medium (DMEM, Sigma). All cell culture media was supplemented with 10% fetal bovine serum (Sigma). Cells were grown in an atmosphere of 21% oxygen and 5% CO2 at 37 ⁇ C.
- Incucyte analysis A panel of breast cancer cell lines, including MCF7 (hormone positive) and MDA-MB-231 (triple-negative breast cancer) were evaluated to examine the impact of Drpin on cellular proliferation. All cell lines were maintained in DMEM medium supplemented with 10% FBS. All cell lines were cultured at 37°C in a humidified 5% CO 2 atmosphere.
- Lipofectamine RNAimax (Life technologies) was used to aid Drpin delivery.
- 2.5 x 10 3 cells were seeded into clear walled 96-well plates and four regions per well were imaged every two hours over a period of 120 h using the Incucyte Zoom (Essen Biosciences). Image analysis was conducted using the Incucyte Zoom software package. Flow cytometry Cell pellets were created by spinning at 11,000rpm for 3min in eppendorf tubes and removing the supernatant. Pellets were resuspended in 180 ⁇ l of 70% ethanol and stored at 4°C overnight.
- the cells were re-pelleted at 14,000g 1min, S/N removed and each sample resuspended in 200 ⁇ l PI solution (2.2.1) with Rnase A added 1:100v/v (100 ⁇ g/ml final). Analyse on FACS 2D histogram for PI. Results To determine if the Drpin could bind to cMyc and cMyc:Max, the inventors conducted a EMSA assay with increasing concentrations of Drpin. It was demonstrated that the Drpin binds to cMyc alone and the cMyc:Max dimer with an IC50 of 46 nM and 107 nM respectively ( Figure 2).
- TNBC triple negative breast cancer
- Example 2 Example 1 confirmed that DrpinMyc was capable of arresting cancer cell line growth at a number of different stages of the cell cycle. However, this did not prove on-target activity for this oligonucleotide.
- the inventors treated MCF7 and MDA- MB-231 cells with DrpinMyc or with a negative RNA sequence of the same chemistry and investigated gene expression profiles of these respective treatment groups. Materials & methods Cell treatment MCF7 and MDA-MB-231breast cancer cell lines (“NEG”) were treated with vehicle (“MOCK”) or DrpinMyc (“DR”).
- RNAseq Each dataset was processed individually for sequencing quality using FastQC. Upon passing quality criteria, reads were aligned against Human genome using STAR aligner. Subsequently, gene and transcript level abundance were estimated with RSEM tool, followed by differential expression analysis using DESeq2 R package. Gene level summarization counts were normalized to library size and batch-effects were corrected for unbiased comparison. Significant differentially expressing genes were identified with significance scores calculated and provided separately for further analysis.
- Raw data processing & read counting Prior to differential expression (DE) analysis, raw reads were evaluated for quality check in terms of sequencing quality, contamination and over-representation of sequences. Reads were then aligned to latest reference genome (GRCh38) with GTF from Gencode (v39) using STAR aligner (Dobin et al, 2013). Post alignment, efforts have been made to utilise the unmapped reads by trimming to dynamic minimum of 36bp length for realignment. Aligned reads were transformed into read counts per gene/transcript using RSEM tool (Li et al, 2011). Differential expression Pairwise differential expression analysis was performed using DESeq2 R package (Love et al, 2014) with Control/Untreated samples being reference for individual experiments.
- Results are split for up-and down-regulated genes (Table 1). Results The present Example revealed a number of genes were depleted in the DrpinMyc treated TNBC cells as compared to the mock treated cells ( Figures 8 & 9). This RNAseq data further demonstrated that of the top twenty repressed transcripts with DrpinMyc treatment, 11 were known targets of Myc, 4 were known to regulate Myc and one was a target of another E-box transcription factor. The remainder have no known regulator at this time. The main repressed transcription factors were: E2F4, FOXM1, SUZ12 and EZH2. All are direct targets of c-Myc, except E2F4, which functions with Myc to drive certain genes.
- tumours reached an average size of 50 mm 3
- animals were treated with DRpin-Myc delivered by tail vein injection (1 injection per week for 6 weeks).
- Four doses of DRpin-Myc were tested (0, 1, 2.5, 5 mg/kg, 8 animals per dose).
- Tumour volume change (%) (tumour volume Day x – tumour volume Day 0) / tumour volume Day 0 x 100).
- TNBC MDA-MB-231 xenograft model Seven week old female NOD-scid gamma (NSG) mice were injected (3 rd mammary fat pad) with 2.5 x 10 6 MDA-MB-231 cells.
- tumours reached an average size of 50 mm 3
- animals were treated with DRpin-Myc delivered by retro-orbital injection (2 injections per week for 4 weeks). Two doses of DRpin-Myc were tested (5 and 10 mg/kg, 6 animals per dose).
- Tumour volume change (%) (tumour volume Day x – tumour volume Day 0) / tumour volume Day 0 x 100).
- Results A dose of 5 mg/kg of DRpin-Myc significantly reduced tumour growth in the TNBC PDX mouse model, without affecting weight gain in treated animals.
- Example 4 This Example was performed to develop a model of DRpinMYC and the high-affinity DNA consensus sequence bound to the MYC:MAX complex and provide further data to develop the DRpin platform for controlled selectivity against transcription factors. Methods DRpinMYC model generation. The 2D structure dot-bracket notation was generated through the MC-Fold web server with default settings (Parisien & Major, 2008). The provided DRpinMYC hairpin sequence was 5'- GAGCACGUGGUUAAAAAACCACGUGCUC -3' (SEQ ID NO: 1).
- DRpinMYC taking the form, 5′- mG*mA*mG*mC*mA*mC*mG*mU*mG*mG*mU*mU*mA*mA*mA*mA*mA*mA*mC *mC*mA*mC*mG*mU*mG*mC-3′.
- Conversion of the RNAComposer output to the RPPS and SPPS conformations was carried out using the alter command in PyMOL from Schrödinger, Inc (Schrödinger, L. & DeLano, W., 2020. PyMOL, Available at: http://www.pymol.org/pymol).
- the model of the high-affinity MYC DNA consensus sequence bound to the MYC:MAX complex was derived by mutating the bases within the duplex of the crystal structure RCSB PDB: 1NKP (Nair & Burley, 2003). Note that the crystal structure contains only the bHLH domains of both proteins. Bound models of DRpinMYC were generated using the MYC: MAX chains of 1NKP. The DNA duplex was removed, and the DRpinMYC models were translated to reproduce an essential interaction between MYC and the core CG of the E-box sequence 5′- CACGUG-3′ (SEQ ID NO: 12) (Nair & Burley, 2003).
- RNA Amber force fields do not include parameters for the PS with 2′-OMe chemistry used herein.
- creating a force field library was necessary to represent the modified form of each nucleotide in both 5', 3' and central residues with both RP and SP PS stereoisomers.
- RESP charges for the library were generated in two runs with RED Server Development (Vanquelef et al., 2011). Partial charges are calculated by constructing the central 2′-OMe nucleotide in the C3′-endo geometry only, as the 2′-OMe is known to enhance this geometry.
- MD system creation Solvent dynamics of oligonucleotide-MYC: MAX systems were studied, and each system was generated with tleap from the AmberTools16 suite (Case et al., 2005). The DNA ⁇ OL15 force field was used to represent the DNA systems (Zgarbova et al., 2013; Zgarbova et al., 2011; Zgarbova et al., 2015). MYC and MAX residues were parameterized with the ff14SB protein force field (Maier et al., 2015).
- Explicit TIP3P water molecules were added to a padding distance of 17 ⁇ in a box, and ionic strength NaCl was added to a concentration of 0.1 M (Mark & Nilsson, 2001). In addition to ionic strength, Na+ ions were added to neutralise the charge. All systems were solvated with ⁇ 33,000 water molecules, 100 Na+ and 80 Cl- ions. Oligonucleotide-MYC: MAX MD simulation All simulations were performed in triplicate using pmemd.cuda from the Amber 2020 package (Case et al., 2005; Salomon-Ferrer, Gotz, Poole, Le Grand, & Walker, 2013). Periodic boundary conditions were imposed in all directions.
- the solvent and counterions were heated from 0 to 300 °K over 50 ns of simulation by applying a 1 kcal/(mol• ⁇ 2) harmonic position restraint to the nucleotide heavy atoms with a constant number, volume and temperature (NVT) ensemble.
- the harmonic restraint was removed, and the system was minimised for 10,000 steps of steepest descent and 5,000 steps of conjugated gradient minimisation.
- the system was equilibrated by heating from 0 to 300 °K over 50 ns under the prior conditions. Production simulations were run at 300 °K using a Langevin thermostat with a collision frequency of 1.0 ps ⁇ 1, constant pressure (NPT) at 1 atm, to a total production time frame of 2 ⁇ s.
- MM/GBSA or generalised Born and surface area continuum solvation
- Born and surface area continuum solvation is a popular relative free energy of binding technique used to determine the strength by which a ligand binds a receptor.
- These energetics are typically calculated from molecular dynamics simulations of the receptor-ligand complex and are more thorough than empirical scoring methods.
- the free energy difference of the binding interaction would be calculated as below (Genheden and Ryde, 2015). Where [A] represents the ligand in solvent and [B] represents the protein. Unfortunately, the energy contribution to binding would come mostly from solvent-solvent interactions, and the total energy would be far larger than the binding energy.
- thermodynamic cycle is broadly defined by the below equation and visualised in Figure 12: ⁇ G solv, complex, ⁇ G solv, receptor, and ⁇ G solv, ligand is solvation free energies of the ligand-protein complex, the protein, and the ligand, respectively. Free energies are estimated using a continuum Poisson–Boltzmann/surface area approach (Genheden and Ryde, 2015). Results A larger E-box surface area strengthens DRpinMYC binding. From Figure 13, Each simulated system represents 6 ⁇ s of simulation time. All systems plateaued at ⁇ 3-4 ⁇ within 50 ns; RMSD plots and a clustering summary are provided in Appendix A and B, respectively.
- the high-affinity MYC consensus sequence demonstrates the more compact arrangement of the B-form DNA geometry.
- the DRpinMYC stereoisomers demonstrate the bulkier arrangement of the A-form geometry imparted by the 2′-OMe chemistry.
- Both the MYC and MAX basic region and the first helix of the bHLH domain bend to accommodate the backbone interactions of the bulkier DRpinMYC geometry compared to DNA. This bending is made possible by the loop region of the bHLH domain; this loop is largely disordered, allowing the first helix to pivot and impart more interactions between MYC and DRpinMYC than the DNA duplex.
- Both stereoisomers of DRpinMYC demonstrate a clear decrease of -50 kcal/mol in binding energy and, thus, a far stronger interaction of DRpinMYC compared to the high-affinity DNA sequence.
- the Major driving force behind binding in both cases are electrostatic interactions between backbone PD or PS groups and the basic amino acids of the first helix of MYC and MAX.
- Evdw values demonstrate the greatest change in an individual value of -24 kcal/mol and align with the increased buried surface area in DRpinMYC systems.
- Esurf due to a greater surface area with which solvent-solute interaction may occur and is imparted by the bulkier DRpinMYC molecule.
- the increased buried surface area seen in Figure 13 imparts stronger or more Van Der Waal interactions between ligand and complex when compared to DNA, while the larger surface area also imparts more solvent-solute interactions.
- the pairwise interactions of the DNA duplex and the DRpinMYC stereoisomers are highly similar, as seen in Figure 14. There is little change in the MYC or MAX residues involved in binding, and electrostatic interactions between basic amino acids, such as Arginine or Lysine, and the oligonucleotide backbone still dominate. Some base interactions between MYC: MAX and DRpinMYC have shifted by 1-2 bases, and all interactions are more distributed than in the DNA duplex.
- the 5′ segment of the DRpinMYC hairpin represents the majority of interactions with MYC, while the 3′ segment interacts with MAX.
- the loop of DRpinMYC and the 5′ bases anterior to the E-box also contribute to the binding.
- the binding energy decomposition indicates that a similar complex is formed in both DRpinMYC and high-affinity DNA interactions. Discussion For this study, DRpinMYC stereoisomers were manually docked to the MYC: MAX complex, then solvated, simulated for several ⁇ s and compared to their DNA counterpart. Systems were analysed by K-means clustering, MM/GBSA and RMSD calculations.
- the data presented herein indicates that the MYC: MAX complex can bind to an A-form PS-linked hairpin of its high-affinity consensus sequence with a greater affinity than its DNA counterpart.
- the geometry of the A-form DRpinMYC and the B-form high-affinity DNA duplex differs.
- MM/GBSA calculations indicate that the MYC: MAX complex binds to DRpinMYC with a stronger affinity than the MYC DNA ligand. Van Der Waals interactions drive this increased affinity due to an increased major groove surface area. Although these interactions are more distributed, as indicated by energy decomposition, the simulation trajectories and clustering indicate that MYC is entirely capable of structural rearrangement to efficiently bind to an A- form geometry.
- R.E.D. Server a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments. Nucleic Acids Res, 39(Web Server issue), W511-517. doi:10.1093/nar/gkr288 Walhout, A. J., Gubbels, J. M., Bernards, R., van der Vliet, P. C., & Timmers, H. T. (1997).
- c- Myc/Max heterodimers bind cooperatively to the E-box sequences located in the first intron of the rat ornithine decarboxylase (ODC) gene.
- ODC rat ornithine decarboxylase
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Animal Behavior & Ethology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The present disclosure relates to the field of oligonucleotides. More particularly, the present disclosure relates to hairpin oligonucleotides for the specific binding and inhibition of target proteins, such as transcription factors. Additionally, this disclosure relates to methods of using such oligonucleotides in preventing or treating diseases, disorders or conditions associated with such target proteins.
Description
"Hairpin Oligonucleotides" Cross-reference to related applications The present application claims priority from Australian Provisional Patent Application No. 2022902346 filed on 17 August 2022, the contents of which is incorporated herein by reference in its entirety. Technical field The present disclosure relates to the field of oligonucleotides. More particularly, the present disclosure relates to hairpin oligonucleotides for the specific binding and inhibition of target proteins, such as transcription factors. Additionally, this disclosure relates to methods of using such oligonucleotides in preventing or treating diseases, disorders or conditions associated with such target proteins. Background Nucleic acid binding proteins, such as transcription factors, can play a role at various points in cell signalling pathways, so as to modulate many normal cellular processes, such as cell growth and proliferation, metabolism, apoptosis, immune responses, and differentiation. Given these cellular effects, their activity is often found to be deregulated in disease, such as cancer. As such, targeting this class of proteins is a major focus of interest, but the structural disorder and lack of binding pockets have made the rational design of small molecule inhibitors for transcription factors challenging. Accordingly, there remains a need for new inhibitors of nucleic acid binding proteins, and particularly those that can specifically target transcription factor activity. Summary The present disclosure is based on the surprising finding that a hairpin oligonucleotide that includes a consensus cMyc binding site in its stem region can specifically bind to this transcription factor with high affinity and inhibit its proliferative effects in cancer cells. Such an inhibition strategy may be applied to other transcription factors that bind to specific nucleic acid sequences, structures or motifs with high affinity.
In a first aspect, the present disclosure provides a hairpin oligonucleotide for inhibiting a target protein, the hairpin oligonucleotide comprising a loop region and a stem region, wherein the stem region comprises a binding site for the target protein. Suitably, the target protein is a transcription factor, such as an E-box transcription factor. In some examples, the transcription factor is selected from the group consisting of Myc, Myc/Max, Fos/Jun, HIF1α/β, MITF, MyoD, HES family, Hey family, ID1/2/3, E2 family, Twist, AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATF1, ATF2, ATF4, ATF5, ATF6, ATF7, ATOH1, ATOH7, ATOH8, BACH1, BACH2, BATF, BATF2, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, CREB1, CREB3, CREB3L1, CREB3L2, CREB3L3, CREB3L4, CREB5, CREBL1, CREM, E4BP4, EPAS1, FERD3L, FIGLA, FOSL1, FOSL2, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, JUN, JUNB, JUND, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MXD3, MXD4, MXI1, MYC, MYCL1, MYCL2, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NFE2, NFE2L2, NFE2L3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, OAF1, OLIG1, OLIG2, OLIG3, OPAQUE2, PTF1A, SCL, SCXB, SIM1, SIM2, SNFT, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL2, TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1, USF2 and any combination thereof. In one example, the transcription factor is Myc or Myc/Max. Suitably, the binding site comprises, consists of or consists essentially of the nucleic acid sequence of 5'-CAC[GA]TG-3', 5'-CAC[GA]UG-3' or a fragment, variant or derivative thereof. In certain examples, the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-GAGCACGUGGUU-3' (SEQ ID NO: 3) or a fragment, variant or derivative thereof and wherein one or more of the U nucleotides thereof may be a T. Suitably, the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleotide sequence of SEQ ID NO: 1 or a fragment, variant or derivative thereof.
In some examples, the stem region comprises a sense strand and an antisense strand, each strand having 5 to 50 nucleotides. In other examples, the hairpin oligonucleotide comprises one or a plurality of phosphorothioate internucleotide linkages. Suitably, the hairpin oligonucleotide comprises one or a plurality of modified nucleotides. In certain examples, the one or plurality of modified nucleotides comprise a modification selected from the group consisting of LNA, HNA, CeNA, 2’-methoxyethyl, 2’-O-alkyl, 2’-O-allyl, 2’- C- allyl, 2’-fluoro, 2’-deoxy, and combinations thereof. In various examples, one or more of the modified nucleotides are modified with 2’-OCH3. In a second aspect, the present disclosure provides a pharmaceutical composition comprising the hairpin oligonucleotide of the first aspect and a pharmaceutically-acceptable carrier, diluent or excipient. In a third aspect, the present disclosure relates to a method of inhibiting a target protein of a cell, said method including the step of contacting the cell with the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect. In a fourth aspect, the present disclosure provides for the use of the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect in the manufacture of a medicament for inhibiting a target protein of a cell. In a fifth aspect, the present disclosure resides in a method of preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject, said method including the step of administering to the subject a therapeutically effective amount of the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect to thereby prevent, ameliorate or treat the disease, disorder or condition. In a sixth aspect, the present disclosure relates to the use of the hairpin oligonucleotide of the first aspect or the pharmaceutical composition of the second aspect in the manufacture of a medicament for preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject.
Referring to the fifth and sixth aspects, the disease, disorder or condition suitably is or comprises a cancer. In some examples, the cancer is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, cancer of the brain and nervous system, head and neck cancer, colon cancer, colorectal cancer, gastric cancer, liver cancer, kidney cancer, melanoma, skin carcinoma, lymphoid cancer, myelomonocytic cancer, pancreatic cancer, pituitary cancer, bone cancer and soft tissue cancer. In one example, the cancer is or comprises breast cancer. In a seventh aspect, the present disclosure provides a method for selecting a hairpin oligonucleotide for inhibiting a target protein, said method including the steps of: (a) producing one or more candidate hairpin oligonucleotides comprising a loop region and a stem region, wherein the stem region comprises a binding site for the target protein; (b) testing the ability of the one or more candidate hairpin oligonucleotides to inhibit the target protein, and (c) selecting a hairpin oligonucleotide which binds and/or inhibits the target protein. In an eighth aspect, the present disclosure relates to a hairpin oligonucleotide designed, selected or identified by the method of the seventh aspect. Brief description of the drawings The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. Figure 1: DRpinMYC forms a stable A-form hairpin. (A) Solution structure of the top cluster of DRpinMYC from a 1 μs atomistic molecular dynamics simulation. Solvent not shown. Bases are labelled according to the sequence's 5' to 3' position. (B) Diagrammatic secondary structure representation of DRpinMYC from MC-Fold composed of twelve Watson-Crick base pairs
with a four-base poly-Adenosine loop and blunt ends. (C) MC-Fold generated dot-bracket notation of secondary structure of DRpinMYC. Scoring indicates a highly stable structure. Figure 2: Binding of DRpin to cMyc:Max dimer. (Left panel) Graph of densitometry of cMyc binding to DRpin-Myc; (Right panel) Graph of densitometry of the Myc:Max dimer binding to DRpin-Myc. NegC is an oligonucleotide of the same chemistry, but lacking the Myc consensus sequence (random sequence). Y axis is relative DRpin-Myc bound by Myc or Myc:Max. X axis is nM of Myc or Myc:Max. Figure 3: A diagram depicting the proposed binding of DRpinMyc with cMyc:Max. Modelling is consistent with the EMSA assays. It suggests the self folding RNA DRpin-Myc molecule can be bound by the cMyc:Max heterodimer. Figure 4: DRpinMyc was used to treat a number of breast cancer cell lines. The drug was either used after resuspension with no additional input other than the buffer or the dissolved drug was heated to 90oC and allowed to cool over 1 hours. This was to determine if DRpinMyc was indeed self folding with no intervention or if it would need an annealing stage. The annealed experiments are labelled with an A (e.g., D2-A). Figure 5: Incucyte analysis of cell growth rate when treated with redissolved DRpinMyc or when treated with re-annealed DRpinMyc. As can be observed DRpinMyc inhibits cell expansion and growth at varying concentrations depending on the cell line. This is consistent with a siMyc treated cells, which arrest in the cell cycle. Figure 6: Cell cycle analysis was performed by flow cytometry using the PI stain. It indicated that like siRNA deletion of cMyc that the cell arrested at different stages of the cell cycle. A represents a 48 hour time point and B a 120 hour time points. The later time points showed that cells were beginning to die. Figure 7: Venn diagram representation of DE genes (fold change beyond +1 and Padj < 0.05) across all contrasts. Figure 8: Heatmap of upregulated transcription factors: Shown are the significantly upregulated transcription factors. The colour scale bar shows z-score values after z-score row normalization. Heatmap was generated using pheatmap package from R. Figure 9: Heatmap of down regulated transcription factors: Shown are the significantly down regulated transcription factors. The colour scale bar shows z-score values after z-score row normalization. Heatmap was generated using pheatmap package from R. Figure 10: DRpin-Myc significantly inhibited TNBC PDX tumour growth. Figure 11: DRpin-Myc significantly inhibited tumour growth in an MDA-MB-231-based xenograft model.
Figure 12: MM/PBSA thermodynamic cycle. MM/PBSA thermodynamic cycle of protein in green and ligand in yellow. Adapted from the Amber Tools 16 manual. Figure 13: Top clusters of oligonucleotides and the MYC: MAX complex. The top representative cluster of each system, derived from 2 μs simulations performed in triplicate. Each system represents a total 6 μs. Myc and Max bHLH/LZ domains are indicated by purple and pink ribbon structures, respectively. A green and yellow ladder structure indicates the DNA duplex. A green ladder structure alone indicates DRpinMYC stereoisomers. The red ladder structure indicates the E-box duplex in each system. Representations of top clusters proportion, cluster RMSD and buried SASA between the complex and DRpinMYC are provided below. Figure 14: MM/GBSA energy decomposition of MYC: MAX systems. Decomposition heatmaps from oligonucleotide, MYC: MAX trajectories. Energies are derived from MM/GBSA calculations and represent 6 μs total per system. MYC, MAX and oligonucleotide 5′, 3′ E-box and loops are marked accordingly. Protein end residues that contributed >-0.2 kcal/mol to binding energies were removed. All energies are presented in kcal/mol and indicated per the colour bar. Key to the Sequence Listing SEQ ID NO: 1 Nucleotide sequence of DRpinMyc in Figure 1 SEQ ID NO: 2 Amino acid sequence of cMyc protein SEQ ID NO: 3 Nucleotide sequence of binding site in DRpinMyc SEQ ID NO: 4 Nucleotide sequence of consensus E-box binding site SEQ ID NO: 5 Nucleotide sequence of exemplary E-box binding site #1 SEQ ID NO: 6 Nucleotide sequence of exemplary E-box binding site #2 SEQ ID NO: 7 Nucleotide sequence of exemplary E-box binding site #3 SEQ ID NO: 8 Nucleotide sequence of non-canonical E-box binding site #1 SEQ ID NO: 9 Nucleotide sequence of non-canonical E-box binding site #2 SEQ ID NO: 10 Nucleotide sequence of non-canonical E-box binding site #3 SEQ ID NO: 11 Nucleotide sequence of exemplary E-box binding site #4 SEQ ID NO: 12 Nucleotide sequence of exemplary E-box binding site #5 SEQ ID NO: 13 Nucleotide sequence of exemplary E-box binding site #6 SEQ ID NO: 14 Nucleotide sequence of consensus E-box binding site #2 SEQ ID NO: 15 Nucleotide sequence of non-canonical E-box binding site #4 SEQ ID NO: 16 Nucleotide sequence of non-canonical E-box binding site #5
SEQ ID NO: 17 Nucleotide sequence of non-canonical E-box binding site #6 SEQ ID NO: 18 Nucleotide sequence of non-canonical E-box binding site #7 SEQ ID NO: 19 Nucleotide sequence of non-canonical E-box binding site #8 SEQ ID NO: 20 Nucleotide sequence of non-canonical E-box binding site #9 SEQ ID NO: 21 Nucleotide sequence of non-canonical E-box binding site #10 SEQ ID NO: 22 Nucleotide sequence of non-canonical E-box binding site #11 SEQ ID NO: 23 Nucleotide sequence of non-canonical E-box binding site #12 SEQ ID NO: 24 Nucleotide sequence of binding site in DRpinMyc variant #1 SEQ ID NO: 25 Nucleotide sequence of binding site in DRpinMyc variant #2 SEQ ID NO: 26 Nucleotide sequence of binding site in DRpinMyc variant #3 SEQ ID NO: 27 Nucleotide sequence of binding site in DRpinMyc variant #4 SEQ ID NO: 28 Nucleotide sequence of binding site in DRpinMyc variant #5 SEQ ID NO: 29 Nucleotide sequence of binding site in DRpinMyc variant #6 SEQ ID NO: 30 Nucleotide sequence of binding site in DRpinMyc variant #7 SEQ ID NO: 31 Nucleotide sequence of binding site in DRpinMyc variant #8 SEQ ID NO: 32 Nucleotide sequence of binding site in DRpinMyc variant #9 Detailed description General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology). The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA technology and immunology. Such procedures are described, for example in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, Second Edition., 1995), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83-115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text;
Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A Practical Guide to Molecular Cloning (1984) and Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series. Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein. Each feature of any particular aspect or embodiment or embodiment of the present disclosure may be applied mutatis mutandis to any other aspect or embodiment or embodiment of the present disclosure. Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter. As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise. For example, a reference to “a bacterium” includes a plurality of such bacteria, and a reference to “an allergen” is a reference to one or more allergens. The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning. As used herein, the term about, unless stated to the contrary, refers to +/- 10%, more preferably
+/-5%, even more preferably +/-1%, of the designated value. Throughout this specification, the word “comprise’ or variations such as “comprises” or “comprising” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. By “consisting essentially of”, in the context of: (a) an amino acid sequence, is meant the recited amino acid sequence together with an additional one, two or three amino acids at the N- and/or C-terminus thereof; or (b) a nucleic acid sequence, is meant the recited nucleic acid sequence together with an additional one, two or three nucleic acids at the 5’ and/or 3’ thereof. All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference. For the present disclosure, the database accession number or unique identifier provided herein for a gene or protein, as well as the gene and/or protein sequence or sequences associated therewith, are incorporated by reference herein. Hairpin oligonucleotides The inventors has surprisingly shown that hairpin oligonucleotides that incorporate a consensus binding site for a target nucleic acid binding protein can be utilised to bind and functionally deplete the target protein and thereby inhibit a biological activity thereof, such as cell growth or proliferation. Accordingly, in one form, there is provided herein a hairpin oligonucleotide for inhibiting a target protein, the hairpin oligonucleotide comprising a loop region and a stem region, wherein the stem region comprises a binding or recognition site for the target protein. In the context of this disclosure, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), wherein the polymer or oligomer of nucleotide monomers contains any combination of nucleobases (referred to in the art and herein as simply as “base”), modified nucleobases, sugars, modified sugars, phosphate bridges, or modified phosphorus atom bridges (also referred to herein as “internucleotide linkage”).
Oligonucleotides can be single-stranded or double-stranded or a combination thereof. A single- stranded oligonucleotide can have double-stranded regions and a double-stranded oligonucleotide can have single-stranded regions (such as a microRNA or shRNA). The terms “hairpin oligo” or “hairpin oligonucleotide” are used interchangeably herein and refer to a nucleic acid molecule, which has a segment (also known as a “hairpin loop” or “loop region”) that is not hybridized with or complementary to a segment of the same hairpin oligonucleotide, and a double-stranded linear region (also known as a “stem” or “duplex” region) having segments that are complementary to each other in the same molecule. Accordingly, the hairpin oligonucleotide suitably comprises a single-stranded loop region that is positioned between a first self-complementary region (e.g., a sense strand) and a second self- complementary region (e.g., an antisense strand) that define, at least in part, the stem region. The hairpin structure of the oligonucleotides described herein is advantageous, as a double stranded oligo of the same chemistry, once entering cells, would have the propensity to equilibrate between corresponding double stranded and single stranded nucleotide structures. Once no longer hybridized in a double stranded manner, a single stranded nucleic acid would further likely not re-anneal to its complementary single stranded nucleic acid due to the dilution factor within a cell. A hairpin oligonucleotide that melts, denatures or dissociates will still maintain a localised high concentration of the respective complementary single stranded stem sequences, which would favour reannealing and formation of the double stranded stem region. In addition, double stranded oligonucleotides that melt, denature or dissociate will have the potential to anneal to homologous RNA and DNA molecules giving the potential for off target activity and/or the mopping up of the single stranded portions of the oligonucleotide preventing them from reannealing to form a double stranded structure. Conversely, the hairpin oligonucleotide provided herein would be less likely to bind/hybridise and/or interact with other off-target nucleic acids, because: (a) the corresponding sequence with the greatest homology is within the same molecule; and (b) the size of the hairpin oligonucleotide reduces its propensity to bind to short complementary nucleic acids. Oligonucleotides generally refer to relatively short sequences of nucleotides, typically with twenty or fewer bases (or nucleotide units), but can also be significantly longer, such as up to about 160 to about 200 nucleotides. By way of example, the hairpin oligonucleotide provided herein is at least about 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100 nucleotides, or any range therein, in length as a single stranded molecule. More particularly, the hairpin oligonucleotide is suitably about 3 to about 75 nucleotides (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 65, 70, or 75 nucleotides, or any range therein) in length as a single stranded molecule. In various examples, the hairpin oligonucleotide is about 15 to about 56, more particularly about 20 to about 40, even more particularly about 25 to about 35, nucleotides in length as a single stranded molecule. In some examples, the hairpin oligonucleotide is about 28 nucleotides in length as a single stranded molecule. Suitably, the stem region is of sufficient length to incorporate a binding site for the target protein. To this end, the stem region (i.e., the double stranded linear or duplex region) provided herein is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50 nucleotide pairs, or any range therein, in length. More particularly, the stem region can be about 6 to about 20 nucleotide pairs in length. Even more particularly, the stem region can be about 8 to about 15 nucleotide pairs in length. Yet even more particularly, the stem region can be about 10 to about 14 nucleotide pairs in length. In certain examples, the stem region is about 12 nucleotide pairs in length. According to various examples, the stem region provided herein is stable (i.e., the self- complementary regions are capable of remaining hybridized together) at approximately 37°C, and unhybridize (i.e., denature) at temperatures greater than 50°C. In other examples, the stem region of the hairpin oligonucleotide has a Gibbs free energy (AG) of unfolding under physiological conditions ranging from about -10 kcal/mol to about -50 kcal/mol (e.g., about - 10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30, -31, -32, -33, -34, -35, -36, -37, -38, -39, -40, -41, -42, -43, -44, -45, -46, -47, -48, -49 or -50 kcal/mol and any range therein). In some examples, the stem region provided herein has a Gibbs free energy (AG) of unfolding under physiological conditions in the range of about -20 kcal/mol to about -40 kcal/mol. In various examples, the stem region provided herein has a Gibbs free energy (AG) of unfolding under physiological conditions of at least about -20 kcal/mol, at least about -25 kcal/mol, at least about -30 kcal/mol or at least about -35 kcal/mol.
The term “loop region” refers to a single-stranded region of more than one nucleotide or modified nucleotide that is not base-paired. Suitably, the loop region is of length sufficient to enable formation of the hairpin structure and base pairing of the stem region. According to particular examples, the loop region provided herein is at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides, or any range therein, in length. More particularly, the loop region can be about 3 to about 10 nucleotides in length. Even more particularly, the loop region can be about 4 to about 6 nucleotides in length. In certain examples, the loop region is about 4 nucleotides in length. It is contemplated that the loop region may include any suitable sequence of nucleotides (e.g., A, C, G, T and/or U). In various examples, the loop region comprises, consists of or consists essentially of a plurality (e.g., 3, 4, 5, 6, 7 etc) of adenine (A) bases. In one example, the loop region comprises, consists of or consists essentially of a nucleotide sequence of 5'-AAAA-3', or a variant or derivative thereof. The hairpin oligonucleotide herein may include single- and/or double-stranded DNA and/or RNA. In some examples, the hairpin oligonucleotide herein (e.g., the hairpin oligonucleotide of SEQ ID NO: 1 or a hairpin oligonucleotide comprising a binding site of SEQ ID NOs: 3-32) includes single-stranded and double-stranded DNA. In other examples, the hairpin oligonucleotide herein (e.g., the hairpin oligonucleotide of SEQ ID NO: 1 or a hairpin oligonucleotide comprising a binding site of SEQ ID NO: 3-32) includes single-stranded and double-stranded RNA. DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and autocatalytic RNA. In this regard, the hairpin oligonucleotide may be a DNA-RNA hybrid. A hairpin oligonucleotide of the present disclosure comprises a nucleotide sequence which typically includes nucleotides that comprise an A, G, C, T or U base. However, nucleotide sequences may include other bases such as inosine, methylycytosine, methylinosine, methyladenosine and/or thiouridine, although without limitation thereto. Contemplated herein are “variant” hairpin oligonucleotides that may include, for example, nucleotide sequences of naturally occurring (e.g., allelic) variants and orthologs (e.g., from a different species) of, for example, a binding site, such as an E-box binding site, inclusive of a consensus sequence thereof. As used herein, a nucleic acid “variant” shares a definable nucleotide sequence relationship with a reference nucleic acid sequence (e.g., a hairpin oligonucleotide, a binding/recognition site, SEQ ID NOs:1, 3-32). The “variant” nucleic acid may have one or a plurality of nucleic acids of the reference nucleic acid sequence deleted or
substituted by different nucleic acids. It is well understood in the art that some nucleic acids of a DNA/RNA-based binding or recognition site may be substituted or deleted without changing (or only having minimal change to) the affinity of the target protein therefor. Suitably, nucleic acid variants share at least 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74% or 75%, more particularly at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more particularly at least 90%, 91%, 92%, 93%, 94%, or 95% nucleotide sequence identity with an isolated nucleic acid of the invention (e.g., SEQ ID NOs: 1, 3 to 32). Percent sequence identity may be determined by any method known in the art, such as that described herein. Terms used generally herein to describe sequence relationships between respective proteins and nucleic acids include “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. Because respective nucleic acids/proteins may each comprise (1) only one or more portions of a complete nucleic acid/protein sequence that are shared by the nucleic acids/proteins, and (2) one or more portions which are divergent between the nucleic acids/proteins, sequence comparisons are typically performed by comparing sequences over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 6, 9 or 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence for optimal alignment of the respective sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (Geneworks program by Intelligenetics; GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA, incorporated herein by reference) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999). The term “sequence identity” is used herein in its broadest sense to include the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard
algorithm, having regard to the extent that sequences are identical over a window of comparison. Thus, a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For example, “sequence identity” may be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA). Also contemplated herein are nucleic acid fragments, such as oligonucleotide fragments. A “fragment” is a segment, domain, portion or region of a nucleic acid, which respectively constitutes less than 100% of the nucleotide sequence. A non-limiting example is an amplification product or a primer or probe. In particular examples, a nucleic acid fragment may comprise, for example, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80 (inclusive of any range therein) contiguous nucleotides of said nucleic acid (e.g., SEQ ID NO: 1). Further contemplated herein are nucleic acid derivatives, inclusive of oligonucleotide derivatives. Such oligonucleotide derivatives may include, for example, one or more modifications and/or conjugates as described herein. By way of example, the hairpin oligonucleotides may be conjugated to one or more moieties or groups which enhance the activity, cellular distribution or cellular uptake thereof. These moieties or groups may be covalently bound to functional groups such as primary or secondary hydroxyl groups. Exemplary moieties or groups include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins and dyes/labels (e.g., Cy5 dye to determine cellular uptake and/or localisation of the hairpin oligonucleotide).
A hairpin oligonucleotide of the present disclosure suitably does not hybridize to a target or known RNA to reduce translation thereof. In particular examples, the hairpin oligonucleotide does not function as an antisense oligonucleotide (i.e., does not comprise a region that is complementary to at least a portion of a specific mRNA molecule, such as encoding an endogenous polypeptide and capable of interfering with a post-transcriptional event, such as mRNA translation). In other examples, the hairpin oligonucleotide is not, or does not form part of, a stranded oligonucleotide for gene silencing (such as RNA interference; i.e., not a double stranded oligonucleotide for siRNA or shRNA). Additionally, in particular examples, the hairpin oligonucleotide is not, or does not function as, an aptamer oligonucleotide. In alternative examples, however, the hairpin oligonucleotides described herein (e.g., the hairpin oligonucleotide of SEQ ID NO: 1 or a hairpin oligonucleotide comprising a binding site of SEQ ID NO: 3-32) may be considered to comprise or function as an aptamer oligonucleotide (e.g., an RNA aptamer or a DNA aptamer). Accordingly, in particular examples, the hairpin oligonucleotide described herein may comprise a synthetic oligonucleotide sequence. As used herein, a “synthetic oligonucleotide sequence” refers to an oligonucleotide sequence which lacks a corresponding sequence that occurs naturally. By way of example, a synthetic oligonucleotide sequence is not complementary to a specific RNA molecule or portion thereof, such as one encoding an endogenous polypeptide. As such, the synthetic oligonucleotide sequence is suitably not capable of directly interfering with a post-transcriptional event, such as RNA translation. Suitably, the hairpin oligonucleotide is capable of inhibiting or reducing an activity of the target protein. As used herein, the phrase “reduces an activity of the target protein” or the like refers to a hairpin oligonucleotide of the present disclosure reducing the ability of a target protein to exert a biological effect. In relation to transcription factors, this may relate to their ability to contact and/or bind to a DNA sequence, such as a promoter sequence or an enhancer sequence, and modulate (e.g., promote or inhibit) expression of the relevant gene. In particular examples, the activity of the target protein, such as contacting and/or binding a DNA sequence, is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of that in the absence of the oligonucleotide.
Inhibition of an activity of the target protein by the hairpin oligonucleotide may be assessed by any means known in the art. In the context of transcription factors, this may be assessed by measuring a level of expression of a target gene, whose expression may be at least partly modulated by the target protein, such as at an mRNA and/or protein level (i.e., transcription and/or translation). This can be directly or indirectly achieved by measuring the amount of RNA encoded by the target gene and/or the amount of protein translated from an encoding RNA. In relation to cMyc, this may be assessed by determining an expression level (e.g., mRNA and/or protein) of one or more markers or mediators of the cell cycle (e.g., E2F1, CDK4, CDC25A, p27, p15), apoptosis (e.g., Bax, Mcl-1, Bcl2), cellular proliferation (e.g., MINA53, ID2, PTMA) and/or cellular metabolism (e.g., CAD, LDHA, ODC-1). Additionally, this can be measured indirectly by assessing a level of activation of cell signalling pathways downstream of the target gene or target protein in question. For example, a level of cMyc signalling may be determined by assessing a level of cell cycle activity, apoptotic activity, cellular proliferation and/or cellular metabolism. Typically, a hairpin oligonucleotide of the present disclosure will be synthesized in vitro, such as by chemical synthesis (e.g., solid-phase synthesis). However, in some instances, particularly where modified bases and a modified backbone are not required, they can be expressed in vitro or in vivo in a suitable system, such as by a recombinant virus or cell. Suitably, the hairpin oligonucleotides described herein are isolated. For the purposes of this disclosure, by “isolated” is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material, such as the hairpin oligonucleotides, may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form. The binding site for the target protein may be complementary to or comprise the entirety of a consensus or reference sequence thereof, or a part thereof. It is also envisaged that the binding site for the target protein may include a variant of a consensus or reference sequence thereof. In this regard, the degree of identity of the sequence of the binding site to the consensus nucleic acid sequence or the reference nucleic acid sequence of the binding site should be at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% and any range therein)
and more particularly 95-100%. The hairpin oligonucleotide may of course comprise unrelated sequences, such as at a 5’ and/or 3’ end of the consensus or reference nucleic acid sequence, which may function to stabilize the molecule, such as described herein. Suitably, the hairpin oligonucleotide provided herein specifically binds to the target protein or binds to the target protein with high affinity. In some examples, the hairpin oligonucleotide binds to the target protein at an affinity that is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500%, or any range therein, greater than the affinity of a DNA ligand (e.g., an unmodified double stranded DNA molecule) comprising, consisting of or consisting essentially of the binding site (e.g., a binding site of SEQ ID NOs: 3-32) of the hairpin oligonucleotide for the target protein. As used herein, the term “binds” refers to the interaction of the hairpin oligonucleotide with the target protein (e.g., a transcription factor, such as cMyc) and means that the interaction is dependent upon the presence of a particular structure (e.g., a binding site having a particular nucleic acid sequence) on the hairpin oligonucleotide that is recognised by the target protein. For example, and by virtue of the binding site, the hairpin oligonucleotide recognizes and binds to a specific target protein rather than to molecules or proteins generally. As used herein, the term “specifically binds” shall be taken to mean that the binding interaction between a hairpin oligonucleotide disclosed herein and a target protein described herein (e.g., cMyc) is dependent on detection of the target protein by the hairpin oligonucleotide. Accordingly, the hairpin oligonucleotide preferentially binds or recognizes the target protein even when present in a mixture of other molecules, proteins, nucleic acids or organisms. As used herein, the terms “high affinity” and “relatively high affinity” are used interchangeably herein and refer to a binding affinity between a hairpin oligonucleotide and the target protein of interest with a KD of at least about 10-6 M, more particularly at least about 10-7 M, more particularly at least about 10-8 M, even more particularly at least about 10-9 M and yet even more particularly between about 10-8 M to about 10-10 M. As used herein, the terms “low affinity” and “relatively low affinity” are used interchangeably herein and refer to a binding affinity between a hairpin oligonucleotide and the target protein of interest with a KD of less than about 10-6 M, preferably less than about 10-5 M, more
preferably less than about 10-4 M and even more preferably between about 10-2 M to about 10- 4 M. The determination of such affinity may be conducted under standard competitive binding immunoassay procedures, as are known in the art, such as electrophoretic shift assay (EMSA), ELISA and surface plasmon resonance (SPR). Suitably, the hairpin oligonucleotides of the present disclosure inhibit the binding between the target protein and one or more of its binding sites (e.g., a consensus DNA binding site, such as the DNA binding sites of SEQ ID NOs: 3-32). In some examples, the hairpin oligonucleotide has a half maximal inhibitory concentration (IC50) of about 500 nM or less (e.g., about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, about 100 nM or less, about 50 nM or less, about 25 nM or less, about 10 nM or less, about 1 nM or less, etc.) for inhibiting binding of the target protein (e.g., cMyc or cMyc:Max) to a binding site thereof (e.g., 5'-CACGTG-3' or 5'-GAGCACGTGGTT-3'). In other examples, the hairpin oligonucleotide has a half maximal inhibitory concentration (IC50) of between about 1 nM and about 500 nM (e.g., about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500 nM or any range therein) for inhibiting binding of the target protein (e.g., cMyc or cMyc:Max) to a binding site thereof (e.g., 5'-CACGTG-3' or 5'- GAGCACGTGGTT-3'). In certain examples, the hairpin oligonucleotide has a half maximal inhibitory concentration (IC50) of about 100 nM or less for inhibiting binding of cMyc to a binding site thereof (e.g., 5'-CACGTG-3' or 5'-GAGCACGTGGTT-3'). In certain examples, the hairpin oligonucleotide has a half maximal inhibitory concentration (IC50) of about 150 nM or less for inhibiting binding of cMyc:Max to a binding site thereof (e.g., 5'-CACGTG-3' or 5'- GAGCACGTGGTT-3'). Methods of measuring the ability of a hairpin oligonucleotide to inhibit binding of a target protein (e.g., human cMyc or cMyc:Max dimer) and a binding site thereof (e.g., 5'-CACGTG-3' or 5'-GAGCACGTGGTT-3') are known in the art, including, without limitation, via EMSA, SPR analysis, ELISA assays, and flow cytometry. Modified Bases Hairpin oligonucleotides of the present disclosure may have nucleobase (“base”) modifications or substitutions. Such modifications can advantageously increase the binding specificity of the stem region for the target protein and/or reduce or minimise the immunogenicity or antigenicity of the hairpin oligonucleotide.
In particular examples, one or more bases (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 etc bases inclusive of any range therein) of the oligonucleotide described herein are modified. In certain examples, one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 5’ end of the hairpin oligonucleotide are modified. In other examples, one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 3’ end of the hairpin oligonucleotide are modified. In some examples, all bases of the oligonucleotide described herein are modified. In alternative examples, no bases of the oligonucleotide described herein are modified. In certain examples, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the bases of the hairpin oligonucleotide are modified. Examples of modified bases include oligonucleotides comprising one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O- alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. In one example, the hairpin oligonucleotide comprises one of the following at the 2' position: O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Further examples of modified oligonucleotides include one or more nucleotides comprising one of the following at the 2' position: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. In particular examples, the modification is selected from the group consisting of a 2'-O-methyl, 2'-O-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2- O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino, fluoroarabinonucleotide, threose nucleic acid or 2'-O--(N-methlycarbamate). In some examples, the modified base comprises a
2'-O-methyl, 2'-fluoro, 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'- bridge, 4'-(CH2)2-O-2'-bridge, 2'-amino, fluoroarabinonucleotide, threose nucleic acid, 2'-O-- (N-methlycarbamate) and any combination thereof. Suitably, the modification includes 2'-methoxy (2'-O-CH3 or 2’OMe), that is, an alkoxyalkoxy group. In certain examples, the hairpin oligonucleotide includes one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 etc nucleotides) at a 5’ end and/or a 3’ thereof that are 2’OMe modified. In particular examples, the hairpin oligonucleotide includes at least one nucleotide at a 5’ end and a 3’ thereof that are 2’OMe modified. In some examples, the hairpin oligonucleotide includes at least one nucleotide at a 5’ end thereof that is 2’OMe modified. In other examples, the hairpin oligonucleotide includes at least one nucleotide at a 3’ end thereof that is 2’OMe modified. In other examples, the modification includes 2'-methoxyethoxy (2'-O-CH2CH2OCH3 (also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., 1995). In further examples, the modification includes 2'-dimethylaminooxyethoxy, that is, a O(CH2)2ON(CH3)2 group (also known as 2'-DMAOE), or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O- dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), that is, 2'-O-CH2-O-CH2-N(CH3)2. Other modifications include 2'-aminopropoxy (2'-OCH2CH2CH2NH2), 2'-allyl (2'-CH2-CH=CH2), 2'- O-allyl (2'-O-CH2-CH=CH2) and 2'-fluoro (2'-F). The 2'-modification may be in the arabino (up) position or ribo (down) position. In certain examples, a 2'-arabino modification is 2'-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, US 4,981,957, US 5,118,800, US 5,319,080, US 5,359,044, US 5,393,878, US 5,446,137, US 5,466,786, US 5,514,785, US 5,519,134, US 5,567,811, US 5,576,427, US 5,591,722, US 5,597,909, US 5,610,300, US 5,627,053, US 5,639,873, US 5,646,265, US 5,658,873, US 5,670,633, US 5,792,747, and US 5,700,920.
A further modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2'- hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. In one example, the linkage is a methylene (-CH2-)n group bridging the 2' oxygen atom and the 4' carbon atom, wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226. Modified nucleobases include other synthetic and natural nucleobases such as, for example, 5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- halouracil and cytosine, 5-propynyl (-CC-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3- deazaadenine, m1A(1-methyladenosine); m2A(2-methyladenosine); Am (2’-O- methyladenosine); ms2m6A(2-methylthio-N6-methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A(2-methylthio-N6isopentenyladenosine); io6A(N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A(2-methylthio-N6-(cis- hydroxyisopentenyl)adenosine); g6A(N6-glycinylcarbamoyladenosine); t6A(N6- threonylcarbamoyladenosine); ms2t6A(2-methylthio-N6-threonyl carbamoyladenosine); m6t6A(N6-methyl-N6-threonylcarbamoyladenosine); hn6A(N6- hydroxynorvalylcarbamoyladenosine); ms2hn6A(2-methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p)(2’-O-ribosyladenosine(phosphate)); I (inosine); m1I(1- methylinosine); m1Im(1,2’-O-dimethylinosine); m3C(3-methylcytidine); Cm(2’-O- methylcytidine); s2C(2-thiocytidine); ac4C(N4-acetylcytidine);
(5-formylcytidine); m5Cm(5,2’-O-dimethylcytidine); ac4Cm(N4-acetyl-2’-O-methylcytidine); k2C(lysidine); m1G(1-methylguanosine); m2G(N2-methylguanosine); m7G(7-methylguanosine); Gm(2’-O- methylguanosine); m22G(N2,N2-dimethylguanosine); m2Gm(N2,2’-O-dimethylguanosine); m2 2Gm(N2,N2,2’-O-trimethylguanosine); Gr(p)(2’-O-ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-
cyano-7-deazaguanosine); preQ1 (7-aminomethyl-7-deazaguanosine); G+ (archaeosine); D (dihydrouridine); m5Um(5,2’-O-dimethyluridine); s4U(4-thiouridine); m5s2U(5-methyl-2- thiouridine); s2Um(2-thio-2’-O-methyluridine); acp3U(3-(3-amino-3-carboxypropyl)uridine); ho5U(5-hydroxyuridine); mo5U(5-methoxyuridine); cmo5U(uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U(5-(carboxyhydroxymethyl)uridine)); mchm5U(5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U(5- methoxycarbonylmethyluridine); mcm5Um(5-methoxycarbonylmethyl-2’-O-methyluridine); mcm5s2U(5-methoxycarbonylmethyl-2-thiouridine); nm5s2U(5-aminomethyl-2-thiouridine); mnm5U(5-methylaminomethyluridine); mnm5s2U(5-methylaminomethyl-2-thiouridine); mnm5se2U(5-methylaminomethyl-2-selenouridine); ncm5U(5-carbamoylmethyluridine); ncm5Um(5-carbamoylmethyl-2’-O-methyluridine); cmnm5U(5- carboxymethylaminomethyluridine); cmnm5Um(5-carboxymethylaminomethyl-2’-O- methyluridine); cmnm5s2U(5-carboxymethylaminomethyl-2-thiouridine); m62A(N6,N6- dimethyladenosine); Im(2’-O-methylinosine); m4C(N4-methylcytidine); m4Cm(N4,2’-O- dimethylcytidine); hm5C(5-hydroxymethylcytidine); m3U(3-methyluridine); cm5U(5- carboxymethyluridine); m6Am(N6,2’-O-dimethyladenosine); m62Am (N6,N6,O-2’- trimethyladenosine); m2,7G(N2,7-dimethylguanosine); m2,2,7G(N2,N2,7-trimethylguanosine); m3Um(3,2’-O-dimethyluridine); m5D(5-methyldihydrouridine); f5Cm (5-formyl-2’-O- methylcytidine); m1Gm (1,2’-O-dimethylguanosine); m1Am(1,2’-O-dimethyladenosine); τm5U(5-taurinomethyluridine); τm5s2U(5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2(isowyosine); or ac6A(N6-acetyladenosine). Further modified nucleobases include tricyclic pyrimidines, such as phenoxazine cytidine (1H- pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), and phenothiazine cytidine (1H-pyrimido[5,4- b][1,4]benzothiazin-2(3H)-one), G-clamps such as, for example, a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), and pyridoindole cytidine (H- pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in US 3,687,808, those disclosed in J.I. Kroschwitz (editor), The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, John Wiley and Sons (1990), those disclosed by Englisch et al.
(1991), and those disclosed by Y.S. Sanghvi, Chapter 15: Antisense Research and Applications, pages 289-302, S.T. Crooke, B. Lebleu (editors), CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the hairpin oligonucleotide. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 oC. In certain examples, these nucleobase substitutions are combined with 2'-O-methoxyethyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, US 3,687,808, US 4,845,205, US 5,130,302, US 5,134,066, US 5,175,273, US 5,367,066, US 5,432,272, US 5,457,187, US 5,459,255, US 5,484,908, US 5,502,177, US 5,525,711, US 5,552,540, US 5,587,469, US 5,594,121, US 5,596,091, US 5,614,617, US 5,645,985, US 5,830,653, US 5,763,588, US 6,005,096, US 5,681,941 and US 5,750,692. Unless stated to the contrary, reference to an A, T, G, U or C base herein can either mean a naturally occurring base or a modified version thereof. Backbones Oligonucleotides of the present disclosure include those having modified backbones or non- natural internucleotide linkages. Oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Such modifications, inclusive of a phosphorothioate backbone, may advantageously protect the hairpin oligonucleotide from digestion by nucleases and/or increase the affinity of the hairpin oligonucleotide to the target protein. This increased affinity may be due, at least in part, to additional electrostatic and London forces. Modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3'-most internucleotide linkage, that is, a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus- containing linkages include, but are not limited to, US 3,687,808, US 4,469,863, US 4,476,301, US 5,023,243, US 5,177,196, US 5,188,897, US 5,264,423, US 5,276,019, US 5,278,302, US 5,286,717, US 5,321,131, US 5,399,676, US 5,405,939, US 5,453,496, US 5,455,233, US 5,466,677, US 5,476,925, US 5,519,126, US 5,536,821, US 5,541,306, US 5,550,111, US 5,563,253, US 5,571,799, US 5,587,361, US 5,194,599, US 5,565,555, US 5,527,899, US 5,721,218, US 5,672,697 and US 5,625,050. Modified oligonucleotide backbones that do not include a phosphorus atom therein include, for example, backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, US 5,034,506, US 5,166,315, US 5,185,444, US 5,214,134, US 5,216,141, US 5,235,033, US 5,264,562, US 5,264,564, US 5,405,938, US 5,434,257, US 5,466,677, US 5,470,967, US 5,489,677, US 5,541,307, US 5,561,225, US 5,596,086, US 5,602,240, US 5,610,289, US 5,602,240, US 5,608,046, US 5,610,289, US 5,618,704, US 5,623,070, US 5,663,312, US 5,633,360, US 5,677,437, US 5,792,608, US 5,646,269 and US 5,677,439.
Suitably, the hairpin oligonucleotide described herein at least partly comprises a modified backbone. Exemplary modified backbones useful for the invention can include those which comprise a phosphorothioate, a non-bridging oxygen atom substituting a sulfur atom, a phosphonate such as a methylphosphonate, a phosphodiester, a phosphoromorpholidate, a phosphoropiperazidate, amides, methylene(methylamino), fromacetal, thioformacetal, a peptide nucleic acid or a phosphoroamidate such as a morpholino phosphorodiamidate (PMO), N3’-P5’ phosphoramidite or thiophosphoroamidite. In particular examples, the oligonucleotide comprises a 5’ region comprising one or more bases which are modified and/or which have a modified backbone and a 3’ region comprising one or more bases which are modified and/or which have a modified backbone. In some examples, all internucleotide linkages of the hairpin oligonucleotide described herein are modified or comprise a modification, such as a phosphorothioate modification. In alternative examples, no internucleotide linkages of the hairpin oligonucleotide described herein are modified or comprise a modification. In particular examples, the hairpin oligonucleotide comprises one or a plurality of phosphorothioate internucleotide linkages. More particularly, all internucleotide linkages of the hairpin oligonucleotide comprise a phosphorothioate modification. In certain examples, at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or any range therein of the internucleotide linkages of the hairpin oligonucleotide are modified or non-natural. In particular examples, at least a portion of the oligonucleotide has/is a ribonucleic acid, deoxyribonucleic acid, a DNA phosphorothioate, an RNA phosphorothioate, 2'-O-methyl- oligonucleotide, 2'-O-methyl-oligodeoxyribonucleotide, 2'-O-hydrocarbyl ribonucleic acid, 2'- O-hydrocarbyl DNA, 2'-O-hydrocarbyl RNA phosphorothioate, 2'-O-hydrocarbyl DNA phosphorothioate, 2'-F-phosphorothioate, 2'-F-phosphodiester, 2'-methoxyethyl phosphorothioate, 2-methoxyethyl phosphodiester, deoxy methylene(methylimino) (deoxy MMI), 2'-O-hydrocarby MMI, deoxy-methylphos-phonate, 2'-O-hydrocarbyl methylphosphonate, morpholino, 4'-thio DNA, 4'-thio RNA, peptide nucleic acid, 3'-amidate, deoxy 3'-amidate, 2'-O-hydrocarbyl 3'-amidate, locked nucleic acid, cyclohexane nucleic acid, tricycle-DNA, 2'fluoro-arabino nucleic acid, N3'-P5' phosphoroamidate, carbamate linked, phosphotriester linked, a nylon backbone modification and any combination thereof. Methods of treating disease associated with a target protein
The present inventors have also surprising shown that the hairpin oligonucleotides described herein can be utilised to bind the target protein, such as a transcription factor, in cells and thereby inhibit a biological activity thereof. As such, the hairpin oligonucleotides may be administered to a subject in need thereof to prevent a disease, disorder or condition associated with or dependent on increased activity and/or expression of the target protein. Therefore, in one form, the present disclosure provides a method of inhibiting a target protein of a cell, said method including the step of contacting the cell with an effective amount the hairpin oligonucleotide described herein. In a related form, the present disclosure provides for the use of the hairpin oligonucleotide or the pharmaceutical composition provided herein in the manufacture of a medicament for inhibiting a target protein of a cell. In another related form, the present disclosure provides a hairpin oligonucleotide or a pharmaceutical composition described herein for use in inhibiting a target protein of a cell. It is contemplated that such methods may be performed in relation to cells in vitro, in vivo and/or ex vivo. In some examples, the method is performed in vitro, such as with cancer cells or patient-derived xenografts isolated from a subject, an organoid or a stem cell-derived (e.g., induced pluripotent stem cell(iPSC)-derived) cell, as described herein. Accordingly, in some examples, the present method includes the earlier step of isolating the cells expressing the target protein from a subject. In other examples, the present method is performed in vivo in a subject. As used herein, the term “effective amount” and the like refers to an amount of the hairpin oligonucleotide or the pharmaceutical composition that is sufficient to induce a desired physiological outcome (e.g., at least partly inhibiting a target protein’s activity). An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period which the individual dosage unit is to be used, the bioavailability of the composition, the route of administration, etc. In another form, the present disclosure provides a method of preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject, said
method including the step of administering to the subject a therapeutically effective amount of the hairpin oligonucleotide or the pharmaceutical composition described herein to thereby prevent, ameliorate or treat the disease, disorder or condition. In a related form, the present disclosure also provides for the use of the hairpin oligonucleotide or the pharmaceutical composition described herein in the manufacture of a medicament for preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject. In a further related form, the present disclosure also provides for a hairpin oligonucleotide or a pharmaceutical composition described herein for use in preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject. With respect to the aspects described herein, the term “subject”, “patient” and “individual” includes, but is not limited to, mammals, inclusive of humans, performance animals (such as horses, camels, greyhounds), livestock (such as cows, sheep, horses) and companion animals (such as cats and dogs). Suitably, the subject is a human. In some examples, the subject is a female human. In other examples, the subject is a male human. As used herein, “treating”, “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a disease, disorder or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan. As used herein, “preventing”, “prevent” or “prevention” refers to a course of action initiated before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.
The term “ameliorating” as used herein refers to any improvement in the disease state of a patient suffering from the disease, disorder or condition described herein by administering to a subject in need thereof a hairpin oligonucleotide according to the present disclosure. Such an improvement may also be seen as a slowing or stopping of the progression of the disease, disorder or condition in the subject. Hairpin oligonucleotides of the present disclosure can be used to target any protein of interest that binds or interacts with a particular nucleic acid sequence, inclusive of secondary, tertiary and quaternary structures formed thereby. The target protein may be extracellularly expressed or may be intracellularly expressed. Typically, the hairpin oligonucleotide is used to modify a trait of an animal, more typically to treat or prevent a disease, disorder or condition. In some examples, the disease, disorder or condition will benefit from inhibition of the target protein following administration of the hairpin oligonucleotide. To this end, the disease, disorder or condition may be at least partly caused by an increased expression and/or activity of the target protein (e.g., increased expression and/or activity of cMyc). Diseases, disorders and conditions, which can be treated or prevented using a hairpin oligonucleotide of the present disclosure include, but are not limited, to cancer (for example breast cancer, ovarian cancer, cancers of the central nervous system, gastrointestinal cancer, bladder cancer, skin cancer, lung cancer, head and neck cancers, haematological and lymphoid cancers, bone cancer) rare genetic diseases, neuromuscular and neurological diseases (for example, spinal muscular atrophy, Amyotrophic Lateral Sclerosis, Duchenne muscular dystrophy, Huntington’s disease, Batten disease, Parkinson’s disease, amyotrophic lateral sclerosis, Ataxia-telangiectasia, cerebral palsy) viruses (for example, cytomegalovirus, hepatitis C virus, Ebola haemorrhagic fever virus, human immunodeficiency virus, coronaviruses), cardiovascular disease (for example, familial hypercholesterolemia, hypertriglyceridemia), autoimmune and inflammatory diseases (for example arthritis, lupus, pouchitis, psoriasis, asthma), and non-alcoholic and alcoholic fatty liver diseases. According to particular examples, the disease, disorder or condition is a cancer. Cancers may include any aggressive or potentially aggressive cancers, tumours or other malignancies such as listed in the NCI Cancer Index at http://www.cancer.gov/types, including all major cancer forms such as sarcomas, carcinomas, lymphomas, leukaemias and blastomas, although without limitation thereto. These may include solid cancers and haematological cancers, and more
particularly breast cancer, lung cancer inclusive of lung adenocarcinoma, cancers of the reproductive system inclusive of ovarian cancer, cervical cancer, uterine cancer and prostate cancer, cancers of the brain and nervous system, head and neck cancers, gastrointestinal cancers inclusive of colon cancer, colorectal cancer and gastric cancer, liver cancer, kidney cancer, skin cancers such as melanoma and skin carcinomas, blood cell cancers inclusive of lymphoid cancers and myelomonocytic cancers, cancers of the endocrine system such as pancreatic cancer and pituitary cancers, musculoskeletal cancers inclusive of bone and soft tissue cancers, although without limitation thereto. It is contemplated that the cancer can be a breast cancer, which may include any aggressive breast cancers and cancer subtypes known in the art, such as triple negative breast cancer, grade 2 breast cancer, grade 3 breast cancer, lymph node positive (LN+) breast cancer, HER2 positive (HER2+) breast cancer, PR negative (PR-) breast cancer, PR positive (PR+) breast cancer, ER negative (ER-) breast cancer and ER positive (ER+) breast cancer. In various examples, the breast cancer is or comprises triple negative breast cancer. The term “therapeutically effective amount” describes a quantity of a specified agent, such as a hairpin oligonucleotide or a composition described herein, sufficient to achieve a desired effect in a subject being treated with that agent or composition. For example, this can be the amount of a hairpin oligonucleotide and optionally one or more further therapeutic agents, necessary to reduce, alleviate and/or prevent a disease, disorder or condition associated with the target protein in question. Suitably, a “therapeutically effective amount” is sufficient to reduce or eliminate a symptom of such a disease, disorder or condition. More particularly, a “therapeutically effective amount” may be an amount sufficient to achieve a desired biological effect, for example an amount that is effective to decrease or prevent disease progression, such as progressive vision loss and blindness. Ideally, a therapeutically effective amount of an agent is an amount sufficient to induce the desired result without causing a substantial cytotoxic effect in the subject. The effective amount of an agent useful for reducing, alleviating and/or preventing the diseases, disorders and conditions described herein will be dependent on the subject being treated, the type and severity of any associated disease, disorder and/or condition (e.g., disease progression), and the manner of administration of the therapeutic composition.
Suitably, the methods described herein further include the step of administering a further therapeutic agent to the subject (i.e., in addition to the hairpin oligonucleotide). As such, the hairpin oligonucleotide may be administered alone (i.e., monotherapy) or alternatively be administered in combination with the further therapeutic agent (e.g., a further anti-cancer agent) which aims to treat or prevent a disease, disorder or condition described herein. In certain examples, the hairpin oligonucleotide described herein may be co-administered with (simultaneously or sequentially) a further treatment of a cancer (e.g., an anti-cancer agent). Non-limiting examples of such treatments include drug therapy, chemotherapy, antibody, nucleic acid and other biomolecular therapies, radiation therapy, surgery, nutritional therapy, relaxation or meditational therapy and other natural or holistic therapies, although without limitation thereto. Generally, drugs, biomolecules (e.g., antibodies, inhibitory nucleic acids such as siRNA) or chemotherapeutic agents are referred to herein as “anti-cancer therapeutic agents” or “anti-cancer agents”. Suitably, the treatment is or comprises one or more of chemotherapy, radiation therapy, molecularly targeted therapy and immunotherapy. As such, in particular examples, the present methods further include the step of administering a therapeutically effective amount of an anti-cancer agent to the subject. The methods of the present disclosure may further include the earlier or initial step of identifying the presence of the disease, disorder or condition associated with the target protein in the subject. Similarly, the methods described herein may include the earlier or initial step of identifying a cancer or a cancer cell as a target protein-dependent cancer or cancer cell. Accordingly, the methods of this disclosure may include the initial step of determining an activity level and/or an expression level of the target protein of a cancer cell or the subject’s cancer, such as from a sample (e.g., a biopsy sample or a biological sample) obtained from the subject or the subject’s cancer. Such a determining step may be performed by any means or method of testing known in the art. Target proteins As used herein, a “target”, such as a “target protein” or “target polypeptide”, refers to a molecule upon which a hairpin oligonucleotide of the invention directly or indirectly exerts its effects (e.g., specifically binds or contacts). Typically, the hairpin oligonucleotide of the present disclosure or a portion thereof and the target protein, or a portion thereof, are able to bind under physiological conditions.
By “protein” is meant an amino acid polymer. The amino acids may be natural or non-natural amino acids, D- or L-amino acids, as are well understood in the art. A “peptide” is generally referred to as a protein typically having no more than fifty (50) amino acids and a polypeptide is generally referred to as a protein typically having more than fifty (50) amino acids. It is envisaged that the target protein may be a nucleic acid binding protein that can specifically bind to a certain nucleic acid sequence. In particular examples, the target protein is a DNA- binding protein. In other examples, the target protein is an RNA-binding protein. In further examples, the target protein is capable of binding both DNA and RNA. Conventional methods for detecting nucleic acid binding proteins, such as transcription factors, include electrophoretic shift assay (EMSA), supershift EMSA, and ELISA-based techniques. A nucleic acid binding protein may be a complex of two or more individual molecules (e.g., cMyc:Max). Such complexes are commonly referred to as “homodimers”, “heterodimers”, “homotype complexes” and “heterotype complexes”. Such a complex suitably comprises a number of individual components joined together by covalent bonds or non-covalent interactions. Given their well-known ability to bind DNA, it is envisaged that the target protein may be a transcription factor that is capable of binding or specifically binding, such as with high affinity, to the binding site of the hairpin oligonucleotide. As such, the hairpin oligonucleotide can inhibit transcription factor function, for example, by interfering with binding thereof to DNA. The term “transcription factor” as used herein means a protein that possesses a biological function that includes regulation, such as initiation or inhibition/repression, of the transcription of one or more genes. That is, a transcription factor is a protein that possesses a DNA-binding domain (DBD) that allows the protein to bind a specific sequence of DNA (e.g., an enhancer element or promoter sequence). Upon binding the enhancer or promoter element, the transcription factor's presence can aid in initiation of transcription by stabilizing transcription initiation complex formation and/or activity, for example. Transcription factors can also inhibit transcription by blocking (i.e., a repressor) the recruitment of RNA polymerase to one or more specific genes, such as by binding to a silencer sequence. Transcription factors also bind to regulatory DNA sequences, such as enhancer sequences, that can be many hundreds of base pairs downstream or upstream from the transcribed gene. Transcription factors can perform the transcription controlling function either alone or in combination with other proteins, such as by
forming an activation complex, and can aid in recruiting RNA polymerase and related proteins to the transcription initiation start site. In view of the above, the binding site of the hairpin oligonucleotide may be or comprise a regulatory DNA sequence, such as a promoter sequence, a silencer sequence or an enhancer sequence, or a fragment or variant thereof. The term “promoter sequence” refers to a polynucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3’-direction) coding sequence. As used herein, the term “enhancer sequence” refers to a sequence capable of increasing gene expression. Such sequences may be located upstream, intronically or downstream of the region to be transcribed. Enhancement of gene expression by enhancer sequences can be achieved through a variety of mechanisms including, but not limited to, increased transcription efficiency, stabilization of mature mRNA, and translation enhancement. The term “silencer sequence” refers to a polynucleotide region comprising a DNA regulatory sequence comprising one or more sequence-specific binding sites for a repressive transcription factor or repressor that can suppress or inhibit transcription from an otherwise active promoter. The binding site of the hairpin oligonucleotide may include one or more additional nucleotides (e.g., 1, 2, 3, 4, 5, etc nucleotides) either side (i.e., 5’ or 3’ end) thereof that are naturally or normally found in a genomic sequence that includes said binding site. Such flanking nucleotide sequences may assist in improving the affinity of the target protein for the binding site. It is further contemplated that the loop region may at least in part facilitate binding of the target protein to the hairpin oligonucleotide. In this regard, the loop region may include a portion, such as a 5’ end or a 3’ end, of the binding site in question. In other examples, the loop region together with the stem region forms a secondary structure that is capable of binding or interacting with a target protein Transcription factors include a wide number of proteins, excluding RNA polymerase, that initiate and regulate the transcription of genes. Exemplary transcription factors include, but are not limited to, AAF, ab1, ADA2, ADA-NF1, AF-1, AFP1, AhR, AIIN3, ALL-1, alpha-CBF, alpha-CP1, alpha-CP2a, alpha-CP2b, alphaHo, alphaH2-alphaH3, Alx-4, aMEF-2, AML1, AML1a, AML1b, AML1c, AML1DeltaN, AML2,
AML3, AML3a, AML3b, AMY-1 L, A-Myb, ANF, AP-1, AP-2alphaA, AP-2alphaB, AP- 2beta, AP-2gamma, AP-3 (1), AP-3 (2), AP-4, AP-5, APC, AR, AREB6, Arnt, Arnt (774 M form), ARP-1, ATBF1-A, ATBF1-B, ATF, ATF-1, ATF-2, ATF-3, ATF-3deltaZIP, ATF-a, ATF-adelta, ATPF1, Barhl1, Barhl2, Barx1, Barx2, Bcl-3, BCL-6, BD73, beta-catenin, Bin1, B-Myb, BP1, BP2, brahma, BRCA1, Brn-3a, Brn-3b, Brn-4, BTEB, BTEB2, B-TFIID, C/EBPalpha, C/EBPbeta, C/EBPdelta, CACCbinding factor, Cart-1, CBF (4), CBF (5), CBP, CCAAT-binding factor, CCMT-binding factor, CCF, CCG1, CCK-1a, CCK-1b, CD28RC, cdk2, cdk9, Cdx-1, CDX2, Cdx-4, CFF, Chx10, CLIM1, CLIM2, CNBP, CoS, COUP, CP1, CP1A, CP1C, CP2, CPBP, CPE binding protein, CREB, CREB-2, CRE-BP1, CRE-BPa, CREMalpha, CRF, Crx, CSBP-1, CTCF, CTF, CTF-1, CTF-2, CTF-3, CTF-5, CTF-7, CUP, CUTL1, Cx, cyclin A, cyclin T1, cyclin T2, cyclin T2a, cyclin T2b, DAP, DAX1, DB1, DBF4, DBP, DbpA, DbpAv, DbpB, DDB, DDB-1, DDB-2, DEF, deltaCREB, deltaMax, DF-1, DF- 2, DF-3, Dlx-1, Dlx-2, Dlx-3, DIx4 (long isoform), Dlx-4 (short isoform, Dlx-5, Dlx-6, DP-1, DP-2, DSIF, DSIF-p14, DSIF-p160, DTF, DUX1, DUX2, DUX3, DUX4, E, E12, E2F, E2F+E4, E2F+p107, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, E2F-6, E47, E4BP4, E4F, E4F1, E4TF2, EAR2, EBP-80, Ebola viral protein 30 (Ebola virus VP30), EC2, EF1, EF-C, EGR1, EGR2, EGR3, EIIaE-A, EIIaE-B, EIIaE-Calpha, EIIaE-Cbeta, EivF, EIf-1, EIk-1, Emx-1, Emx-2, Emx-2, En-1, En-2, ENH-bind. prot., ENKTF-1, EPAS1, epsilonF1, ER, Erg-1, Erg- 2, ERR1, ERR2, ETF, Ets-1, Ets-1 delta Vil, Ets-2, Evx-1, F2F, factor 2, Factor name, FBP, f- EBP, FKBP59, FKHL18, FKHRL1P2, Fli-1, Fos, FOXB1, FOXC1, FOXC2, FOXD1, FOXD2, FOXD3, FOXD4, FOXE1, FOXE3, FOXF1, FOXF2, FOXG1a, FOXG1b, FOXG1c, FOXH1, FOXI1, FOXJ1a, FOXJ1b, FOXJ2 (long isoform), FOXJ2 (short isoform), FOXJ3, FOXK1a, FOXK1b, FOXK1c, FOXL1, FOXM1a, FOXM1b, FOXM1c, FOXN1, FOXN2, FOXN3, FOX01a, FOX01b, FOXO2, FOXO3a, FOXO3b, FOXO4, FOXP1, FOXP3, Fra-1, Fra-2, FTF, FTS, G factor, G6 factor, GABP, GABP-alpha, GABP-beta1, GABP-beta2, GADD 153, GAF, gammaCMT, gammaCAC1, gammaCAC2, GATA-1, GATA-2, GATA-3, GATA- 4, GATA-5, GATA-6, Gbx-1, Gbx-2, GCF, GCMa, GCNS, GF1, GLI, GLI1, GLI3, GR alpha, GR beta, GRF-1, Gsc, Gsc1, GT-IC, GT-IIA, GT-IIBalpha, GT-IIBbeta, H1TF1, H1TF2, H2RIIBP, H4TF-1, H4TF-2, HAND1, HAND2, HB9, HDAC1, HDAC2, HDAC3, hDaxx, heat-induced factor, HEB, HEB1-p67, HEB1-p94, HEF-1 B, HEF-1T, HEF-4C, HEN1, HEN2, Hesx1, Hex, HIF-1, HIF-1alpha, HIF-1beta, HiNF-A, HiNF-B, HINF-C, HINF-D, HiNF-D3, HiNF-E, HiNF-P, HIP1, HIV-EP2, Hlf, HLTF, HLTF (Met123), HLX, HMBP, HMG I, HMG I(Y), HMG Y, HMGI-C, HNF-1A, HNF-1B, HNF-1C, HNF-3, HNF-3alpha, HNF-3beta, HNF-3gamma, HNF4, HNF-4alpha, HNF4alpha1, HNF-4alpha2, HNF-4alpha3, HNF-
4alpha4, HNF4gamma, HNF-6alpha, hnRNP K, HOX11, HOXA1, HOXA10, HOXA10 PL2, HOXA11, HOXA13, HOXA2, HOXA3, HOXA4, HOXA5, HOXA6, HOXA7, HOXA9A, HOXA9B, HOXB-1, HOXB13, HOXB2, HOXB3, HOXB4, HOXB5, HOXB6, HOXA5, HOXB7, HOXB8, HOXB9, HOXC10, HOXC11, HOXC12, HOXC13, HOXC4, HOXC5, HOXC6, HOXC8, HOXC9, HOXD10, HOXD11, HOXD12, HOXD13, HOXD3, HOXD4, HOXD8, HOXD9, Hp55, Hp65, HPX42B, HrpF, HSF, HSF1 (long), HSF1 (short), HSF2, hsp56, Hsp90, IBP-1, ICER-II, ICER-ligamma, ICSBP, Id1, Id1 H′, Id2, Id3, Id3/Heir-1, IF1, IgPE-1, IgPE-2, IgPE-3, IkappaB, IkappaB-alpha, IkappaB-beta, IkappaBR, II-1 RF, IL-6 RE- BP, 11-6 RF, INSAF, IPF1, IRF-1, IRF-2, irlB, IRX2a, Irx-3, Irx-4, ISGF-1, ISGF-3, ISGF3alpha, ISGF-3gamma, 1st-1, ITF, ITF-1, ITF-2, JRF, Jun, JunB, JunD, kappay factor, KBP-1, KER1, KER-1, Kox1, KRF-1, Ku autoantigen, KUP, LBP-1, LBP-1a, LBX1, LCR- F1, LEF-1, LEF-1B, LF-A1, LHX1, LHX2, LHX3a, LHX3b, LHXS, LHX6.1a, LHX6.1b, LIT-1, Lmo1, Lmo2, LMX1A, LMX1B, L-My1 (long form), L-My1 (short form), L-My2, LSF, LXRalpha, LyF-1, LyI-1, M factor, Mad1, MASH-1, Max1, Max2, MAZ, MAZ1, MB67, MBF1, MBF2, MBF3, MBP-1 (1), MBP-1 (2), MBP-2, MDBP, MEF-2, MEF-2B, MEF-2C (433 AA form), MEF-2C (465 AA form), MEF-2C (473 M form), MEF-2C/delta32 (441 AA form), MEF-2D00, MEF-2D0B, MEF-2DA0, MEF-2DA′0, MEF-2DAB, MEF-2DA′B, Meis- 1, Meis-2a, Meis-2b, Meis-2c, Meis-2d, Meis-2e, Meis3, Meox1, Meox1a, Meox2, MHox (K- 2), Mi, MIF-1, Miz-1, MM-1, MOP3, MR, Msx-1, Msx-2, MTB-Zf, MTF-1, mtTF1, Mxi1, Myb, Myc (cMyc), Myc 1, Myf-3, Myf-4, Myf-5, Myf-6, MyoD, MZF-1, NC1, NC2, NCX, NELF, NER1, Net, NF III-a, NF NF NF-1, NF-1A, NF-1B, NF-1X, NF-4FA, NF-4FB, NF- 4FC, NF-A, NF-AB, NFAT-1, NF-AT3, NF-Atc, NF-Atp, NF-Atx, NfbetaA, NF-CLE0a, NF- CLE0b, NFdeltaE3A, NFdeltaE3B, NFdeltaE3C, NFdeltaE4A, NFdeltaE4B, NFdeltaE4C, Nfe, NF-E, NF-E2, NF-E2 p45, NF-E3, NFE-6, NF-Gma, NF-GMb, NF-IL-2A, NF-IL-2B, NF-jun, NF-kappaB, NF-kappaB(-like), NF-kappaB1, NF-kappaB1, precursor, NF-kappaB2, NF-kappaB2 (p49), NF-kappaB2 precursor, NF-kappaE1, NF-kappaE2, NF-kappaE3, NF- MHCIIA, NF-MHCIIB, NF-muE1, NF-muE2, NF-muE3, NF-S, NF-X, NF-X1, NF-X2, NF- X3, NF-Xc, NF-YA, NF-Zc, NF-Zz, NHP-1, NHP-2, NHP3, NHP4, NKX2-5, NKX2B, NKX2C, NKX2G, NKX3A, NKX3A v1, NKX3A v2, NKX3A v3, NKX3A v4, NKX3B, NKX6A, Nmi, N-Myc, N-Oct-2alpha, N-Oct-2beta, N-Oct-3, N-Oct-4, N-Oct-5a, N-Oct-5b, NP-TCII, NR2E3, NR4A2, Nrf1, Nrf-1, Nrf2, NRF-2beta1, NRF-2gamma1, NRL, NRSF form 1, NRSF form 2, NTF, O2, OCA-B, Oct-1, Oct-2, Oct-2.1, Oct-2B, Oct-2C, Oct-4A, Oct4B, Oct-5, Oct-6, Octa-factor, octamer-binding factor, oct-B2, oct-B3, Otx1, Otx2, OZF, p107, p130, p28 modulator, p300, p38erg, p45, p49erg,-p53, p55, p55erg, p65delta, p67, Pax-1, Pax-
2, Pax-3, Pax-3A, Pax-3B, Pax-4, Pax-5, Pax-6, Pax-6/Pd-5a, Pax-7, Pax-8, Pax-8a, Pax-8b, Pax-8c, Pax-8d, Pax-8e, Pax-8f, Pax-9, Pbx-1a, Pbx-1b, Pbx-2, Pbx-3a, Pbx-3b, PC2, PC4, PC5, PEA3, PEBP2alpha, PEBP2beta, Pit-1, PITX1, PITX2, PITX3, PKNOX1, PLZF, PO-B, Pontin52, PPARα, PPARβ, PPARgamma1, PPARγ2, PPUR, PR, PR A, pRb, PRD1-BF1, PRDI-BFc, Prop-1, PSE1, P-TEFb, PTF, PTFα, PTFβ, PTFdelta, PTFγ, Pu box binding factor, Pu box binding factor (BJA-B), PU.1, PuF, Pur factor, R1, R2, RAR-alpha1, RAR-β, RAR-β2, RAR-γ, RAR-γ1, RBP60, RBP-Jκ, Rel, RelA, RelB, RFX, RFX1, RFX2, RFX3, RFXS, RF- Y, RORα1, RORα2, RORα3, RORbeta, RORgamma, Rox, RPF1, RPGα, RREB-1, RSRFC4, RSRFC9, RVF, RXR-α, RXR-β, SAP-1a, SAP1b, SF-1, SHOX2a, SHOX2b, SHOXa, SHOXb, SHP, Sill-p110, SIII-p15, SIII-p18, SIM′, Six-1, Six-2, Six-3, Six-4, Six-5, Six-6, SMAD-1, SMAD-2, SMAD-3, SMAD-4, SMAD-5, SOX-11, SOX-12, Sox-4, Sox-5, SOX-9, Sp1, Sp2, Sp3, Sp4, Sph factor, Spi-B, SPIN, SRCAP, SREBP-1a, SREBP-1b, SREBP-1c, SREBP-2, SRE-ZBP, SRF, SRY, SRP1, Staf-50, STAT1alpha, STAT1beta, STAT2, STAT3, STAT4, STATE, T3R, T3R-α1, T3R-α2, T3R-beta, TAF(I)110, TAF(I)48, TAF(I)63, TAF(II)100, TAF(II)125, TAF(II)135, TAF(II)170, TAF(II)18, TAF(II)20, TAF(II)250, TAF(II)250Delta, TAF(II)28, TAF(II)30, TAF(II)31, TAF(II)55, TAF(II)70-α, TAF(II)70-β, TAF(II)70-γ, TAF-I, TAF-II, TAF-L, Tal-1, Tal-1beta, Tal-2, TAR factor, TBP, TBX1A, TBX1B, TBX2, TBX4, TBXS (long isoform), TBXS (short isoform), TCF, TCF-1, TCF-1A, TCF-1B, TCF-1C, TCF-1D, TCF-1E, TCF-1F, TCF-1G, TCF-2alpha, TCF-3, TCF-4, TCF- 4(K), TCF-4B, TCF-4E, TCFbeta1, TEF-1, TEF-2, tel, TFE3, TFEB, TFIIA, TFIIA-α/β precursor, TFIIA-alpha/beta precursor, TFIIA-gamma, TFIIB, TFIID, TFIIE, TFIIE-α, TFIIE- β, TFIIF, TFIIF-α, TFIIF-β, TFIIH, TFIIH*, TFIIH-CAK, TFIIH-cyclin H, TFIIH- ERCC2/CAK, TFIIH-MAT1, TFIIH-MO15, TFIIH-p34, TFIIH-p44, TFIIH-p62, TFIIH-p80, TFIIH-p90, TFII-I, Tf-LF1, Tf-LF2, TGIF, TGIF2, TGT3, THRA1, TIF2, TLE1, TLX3, TMF, TR2, TR2-11, TR2-9, TR3, TR4, TRAP, TREB-1, TREB-2, TREB-3, TREF1, TREF2, TRF (2), TTF-1, TXRE BP, TxREF, UBF, UBP-1, UEF-1, UEF-2, UEF-3, UEF-4, USF1, USF2, USF2b, Vav, Vax-2, VDR, vHNF-1A, vHNF-1B, vHNF-1C, VITF, WSTF, WT1, WT1I, WT1 I-KTS, WT1 I-del2, WT1-KTS, WT1-del2, X2BP, XBP-1, XW-V, XX, YAF2, YB-1, YEBP, YY1, ZEB, ZF1, ZF2, ZFX, ZHX1, ZIC2, ZID, ZNF174, amongst others. It is contemplated that the transcription factor can be an E-box transcription factor capable of binding an E-box transcription factor binding site or domain (i.e., an enhancer box or E-box). Generally, an E-box is a DNA response element found in some eukaryotes that acts as a protein binding site and has been shown to regulate gene expression in a range of cells and tissues.
Accordingly, the binding site of the hairpin oligonucleotide may comprise, consist of or consist essentially of an E-box binding site, as are known in the art. Suitably, such binding sites comprise, consist of or consist essentially of a nucleotide sequence of 5'-CANNTG-3' (SEQ ID NO: 4; i.e., a consensus E-box binding site sequence), wherein N can be any nucleotide (e.g., A, C, T, G or U). In certain examples, the E-box binding site comprises, consists of or consists essentially of a nucleotide sequence of 5'-CAC[GA]TG-3' (SEQ ID NO: 5) (wherein [GA] or [G/A] indicates a nucleotide variation of G or A at this position) or more particularly 5'- CACGTG-3' (SEQ ID NO: 6) or 5'-CACATG-3' (SEQ ID NO: 7), or a nucleotide sequence having one or two substitutions, deletions, or insertions therein. The substitutions, deletions, or insertions may be any substitution, deletion, or insertion of a single nucleotide such that the E- box transcription factor binding site retains at least one of its endogenous functions (e.g., an ability to bind an E-box transcription factor). By way of example, one or more of the thymine residues may be replaced or substituted by uracil residues or vice versa (e.g., 5'-CANNUG-3' (SEQ ID NO: 14), 5'-CAC[GA]UG-3' (SEQ ID NO: 11), 5'-CACGUG-3' (SEQ ID NO: 12) or 5'-CACAUG-3' (SEQ ID NO: 13)). In addition to the above, non-canonical E-boxes are also known in the art. As such, in alternative examples, the E-box binding site comprises, consists of, consists essentially of a nucleotide sequence of 5'- CACGTT-3' (SEQ ID NO: 8), 5'- CAGCTT-3' (SEQ ID NO: 9) or 5'-CACCTCGTGAC-3' (SEQ ID NO: 10), or a nucleotide sequence having one or two substitutions, deletions, or insertions therein. For such examples and when incorporated into a hairpin oligonucleotide, one or more of the thymine residues may be replaced or substituted by uracil residues or vice versa (e.g., 5'- CACGUU-3' (SEQ ID NO: 15), 5'- CACGUT-3' (SEQ ID NO: 16), 5'- CACGTU-3' (SEQ ID NO: 17), 5'- CAGCUU-3' (SEQ ID NO: 18), 5'- CAGCUT-3' (SEQ ID NO: 19), 5'- CAGCTU-3' (SEQ ID NO: 20), 5'-CACCUCGUGAC-3' (SEQ ID NO: 21), 5'-CACCTCGUGAC-3' (SEQ ID NO: 22) or 5'-CACCUCGTGAC-3' (SEQ ID NO: 23)). Exemplary E-box transcription factors include Myc/Max, Fos/Jun, HIF1α/β, MITF, MyoD, HES family, Hey family, ID1/2/3, E2 family, Twist, AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATF1, ATF2, ATF4, ATF5, ATF6, ATF7, ATOH1, ATOH7, ATOH8, BACH1, BACH2, BATF, BATF2, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, CREB1, CREB3, CREB3L1, CREB3L2,
CREB3L3, CREB3L4, CREB5, CREBL1, CREM, E4BP4, EPAS1, FERD3L, FIGLA, FOSL1, FOSL2, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, JUN, JUNB, JUND, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MXD3, MXD4, MXI1, MYC, MYCL1, MYCL2, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NFE2, NFE2L2, NFE2L3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, OAF1, OLIG1, OLIG2, OLIG3, OPAQUE2, PTF1A, SCL, SCXB, SIM1, SIM2, SNFT, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL2, TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1 and USF2. In particular examples, the target protein is cMyc (inclusive of the cMyc-Max protein complex). cMyc is a protooncogene, which is overexpressed in a wide range of human cancers. When it is specifically mutated, or overexpressed, it increases cell proliferation and functions as an oncogene. The MYC gene encodes for a transcription factor that regulates expression of 15% of all genes through binding on Enhancer Box sequences (E-boxes) and recruiting histone acetyltransferases (HATs). cMyc belongs to the Myc family of transcription factors, which also includes nMyc and lMyc. Myc-family transcription factors contain the bHLH/LZ (basic Helix- Loop-Helix Leucine Zipper) domain. In some examples, cMyc protein or simply cMyc relates to human cMyc. A non-limiting example of a cMyc amino acid sequence from humans may be found under accession number P01106 (UniProtKB). An exemplary amino acid sequence for a full length cMyc is provided in SEQ ID NO: 2 below: cMyc protein (SEQ ID NO:2) MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPP LSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPD DETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQD LSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVL HEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRC HVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHN VLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLL RKRREQLKHKLEQLRNSCA cMyc is known to bind DNA in a non-specific manner, but can also specifically recognize the core sequence of 5'-CAC[GA]TG-3' (SEQ ID NO: 5). Accordingly, the binding site of the hairpin oligonucleotide may comprise, consist of or consist essentially of the nucleic acid sequence of 5'-CAC[GA]TG-3' (SEQ ID NO: 5) or 5'-CAC[GA]UG-3' (SEQ ID NO: 11).
Referring to some examples, the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of a nucleic acid sequence of 5'-CACGTG-3' (SEQ ID NO: 6), 5'- CACGUG-3' (SEQ ID NO: 12), 5'-CACATG-3' (SEQ ID NO: 7), 5'-CACAUG-3' (SEQ ID NO: 13) or a fragment, variant or derivative thereof. In particular examples, the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-CACGTG-3' (SEQ ID NO: 6) or 5'-CACGUG-3' (SEQ ID NO: 12). In other examples, the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-CACATG-3' (SEQ ID NO: 7) or 5'-CACAUG-3' (SEQ ID NO: 13). Suitably, the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-GAGCAC[GA]UGGUU-3' (SEQ ID NO: 31), wherein one or more of the U nucleotides may be a T. In some examples, the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5'-GAGCACGUGGUU-3' (SEQ ID NO: 3) or a fragment, variant or derivative thereof. Such variants may include the nucleotide sequence of 5'-GAGCACAUGGUU-3' (SEQ ID NO: 32), wherein one or more of the U nucleotides may be a T. The nucleotide sequence of SEQ ID NO: 3 represents the NPM1 gene’s E-box promoter sequence inclusive of adjacent, flanking or surrounding base pairs, which is regarded as a high affinity binding site for cMyc. With respect to the binding site of SEQ ID NO:3, it is envisaged that one or more of the U nucleotides may be a T (e.g., 5'-GAGCACGTGGUU-3' (SEQ ID NO: 24), 5'- GAGCACGTGGTU-3' (SEQ ID NO: 25), 5'-GAGCACGTGGTT-3' (SEQ ID NO: 26), 5'- GAGCACGTGGUT-3' (SEQ ID NO: 27), 5'-GAGCACGUGGTU-3' (SEQ ID NO: 28), 5'- GAGCACGUGGUT-3' (SEQ ID NO: 29) or 5'-GAGCACGUGGTT-3' (SEQ ID NO: 30)). Suitably, the hairpin oligonucleotide, in a single stranded form, comprises, consists of or consists essentially of the nucleic acid sequence of 5'- GAGCACGUGGUUAAAAAACCACGUGCUC-3' (SEQ ID NO: 1; Figure 1) or a fragment, variant or derivative thereof. Again, it will be understood that one or more of the U nucleotides of SEQ ID NO: 1 may be a T. It is contemplated that one or more of the nucleotides and/or the internucleotide linkages of SEQ ID NO: 1 can be modified, such as described herein. In particular examples, all of the internucleotide linkages of SEQ ID NO: 1 comprise a phosphorothioate modification. In certain examples, the 3’ terminal cytosine (C) residue of SEQ ID NO: 1 comprises a 2’OMe modification. In other examples, the 5’ terminal guanine
(G) residue of SEQ ID NO: 1 comprises a 2’OMe modification. According to other examples, the 3’ terminal cytosine (C) residue and the 5’ terminal guanine (G) residue of SEQ ID NO: 1 comprises a 2’OMe modification. For particular examples, all of the internucleotide linkages of SEQ ID NO: 1 comprise a phosphorothioate modification and all of the nucleic acid bases of SEQ ID NO: 1 are modified, such as by way of a 2’OMe modification (e.g., 5′- mG*mA*mG*mC*mA*mC*mG*mU*mG*mG*mU*mU*mA*mA*mA*mA*mA*mA*mC *mC*mA*mC*mG*mU*mG*mC*mU*mC-3′; phosphorothioate modified internucleotide linkages are indicated by
and 2′-OMe modified nucleic acid bases are indicated by “m”). It is known that cMyc may function in conjunction with Max. These two transcription factors can form a heterodimer on the promoter of a target gene. There are low and high affinity targets for cMyc:Max and thus cMyc’s expression is typically fine-tuned to only drive activation of specific promoters in the context of cell type. In view of the above, the hairpin oligonucleotide can inhibit Myc/Max activity or function, such as by interfering with Myc/Max binding to DNA (e.g., a E-box transcription factor binding site or another DNA binding site, such as those described herein). As used herein, the phrase “inhibits Myc/Max activity” or variations thereof means that after administration of an hairpin oligonucleotide described herein, such as that of SEQ ID NO: 1, to an animal or a cell, an activity of Myc/Max in the animal or cell, such as the ability of this protein complex to bind E boxes, is abrogated or reduced. In various examples, the activity of Myc/Max is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of the activity in the absence of the hairpin oligonucleotide. Formulation In a particular form, the pharmaceutical compositions described herein comprise an acceptable carrier, diluent or excipient. By “acceptable carrier, diluent or excipient” is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, diluent and excipients well known in the art may be used. These may be selected from a group including sugars, starches, cellulose
and its derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and salts such as mineral acid salts including hydrochlorides, bromides and sulfates, organic acids such as acetates, propionates and malonates, water and pyrogen-free water. A useful reference describing acceptable carriers, diluents and excipients is Remington’s Pharmaceutical Sciences (Mack Publishing Co. N.J. USA, 1991) which is incorporated herein by reference. Hairpin oligonucleotides of the disclosure may be admixed, encapsulated, conjugated (such as fused) or otherwise associated with other molecules, molecule structures or mixtures of compounds, resulting in, for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, US 5,108,921, US 5,354,844, US 5,416,016, US 5,459,127, US 5,521,291, US 5,543,158, US 5,547,932, US 5,583,020, US 5,591,721, US 4,426,330, US 4,534,899, US 5,013,556, US 5,108,921, US 5,213,804, US 5,227,170, US 5,264,221, US 5,356,633, US 5,395,619, US 5,416,016, US 5,417,978, US 5,462,854, US 5,469,854, US 5,512,295, US 5,527,528, US 5,534,259, US 5,543,152, US 5,556,948, US 5,580,575, and US 5,595,756. The hairpin oligonucleotides may be formulated as pharmaceutically acceptable salts, esters, or salts of the esters, or any other compounds which, upon administration are capable of providing (directly or indirectly) the biologically active metabolite. The term “pharmaceutically acceptable salts” as used herein refers to physiologically and pharmaceutically acceptable salts of the oligonucleotide that retain the desired biological activities of the parent compounds and do not impart undesired toxicological effects upon administration. Examples of pharmaceutically acceptable salts and their uses are further described in US 6,287,860. It is further envisaged that the hairpin oligonucleotides provided herein may be prodrugs or pharmaceutically acceptable salts of the prodrugs, or other bioequivalents. The term “prodrugs” as used herein refers to therapeutic agents that are prepared in an inactive form that is converted to an active form (i.e., a drug) upon administration by the action of endogenous enzymes or
other chemicals and/or conditions. In particular, prodrug forms of the oligonucleotide of the disclosure are prepared as SATE [(S acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510, WO 94/26764 and US 5,770,713. A prodrug may, for example, be converted within the body, such as by hydrolysis in the blood, into its active form that has medical effects. Pharmaceutical acceptable prodrugs are described in T. Higuchi and V. Stella, Prodrugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series (1976); "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985; and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987. Those skilled in the art of organic chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". In some examples, the hairpin oligonucleotides of the present disclosure can be complexed with a complexing agent to increase cellular uptake thereof. An example of a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells. The term “cationic lipid” includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells. In general, cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof. Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms. Exemplary straight chain or branched alkyl or alkene groups have six or more carbon atoms. Alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., Cl-, Br-, I-, F-, acetate, trifluoroacetate, sulfate, nitrite, and nitrate. Examples of cationic lipids include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINETM (e.g., LIPOFECTAMINETM 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.). Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium
chloride (DOTMA), N-[1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), 3.beta.-[N--(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,- dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB). Oligonucleotides can also be complexed with, e.g., poly (L-lysine) or avidin and lipids may, or may not, be included in this mixture, e.g., steryl-poly (L-lysine). Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., US 5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al., 1996; Hope et al., 1998). In addition to those listed above, other lipid compositions are also known in the art and include, for example, those taught in US 4,235,871; US 4,501,728; 4,837,028; 4,737,323. In some examples, lipid compositions can further comprise agents (e.g., viral proteins) to enhance lipid-mediated transfections of oligonucleotides. In further examples, N-substituted glycine oligonucleotides (peptoids) can be used to optimize uptake of oligonucleotides into tissues and cells. In various examples, a composition for delivering hairpin oligonucleotides of the present disclosure comprises a peptide having from between about one to about four basic residues. These basic residues can be located, e.g., on the amino terminal, C-terminal, or internal region of the peptide. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine (can also be considered non-polar), asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Apart from the basic amino acids, a majority or all of the other residues of the peptide can be selected from the non-basic amino acids, e.g., amino acids other than lysine, arginine, or histidine. Suitably, a preponderance of neutral amino acids with long neutral side chains are used.
Suitably, the hairpin oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide”. In one example, the composition includes an oligonucleotide and a covalently attached transporting peptide. In further examples, the hairpin oligonucleotide is attached to a targeting moiety such as N- acetylgalactosamine (GalNAc), an antibody, an antibody-like molecule or aptamer (see, for example, Toloue and Ford (2011) and Esposito et al. (2018)). Administration Any safe route of administration may be employed, including oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intra-muscular, intra-dermal, subcutaneous, inhalational, intranasal, intraocular, intraperitoneal, intracerebroventricular, topical, mucosal and transdermal administration, although without limitation thereto. In one example, the oligonucleotide of the disclosure is administered systemically. As used herein “systemic administration” is a route of administration that is either enteral or parenteral. Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, nasal sprays, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting controlled releasing devices designed specifically for this purpose or other forms of implants modified to act additionally in this fashion. Controlled release may be effected by coating with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivatives such as hydroxypropylmethyl cellulose. In addition, the controlled release may be effected by using other polymer matrices, liposomes and/or microspheres. Compositions may be presented as discrete units such as capsules, sachets, functional foods/feeds or tablets each containing a pre-determined amount of one or more therapeutic agents of the disclosure, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into association one or more agents as described above with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by
uniformly and intimately admixing the agents of the disclosure with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. The above compositions may be administered in a manner compatible with the dosage formulation, and in such amount as effective. The dose administered to a subject, in the context of the present disclosure, should be sufficient to effect a beneficial response in a subject over an appropriate period of time. The quantity of agent(s) to be administered may depend on the subject to be treated inclusive of the age, sex, weight and general health condition thereof, factors that will depend on the judgement of the practitioner. Design and Testing of Candidate Oligonucleotides In another form, the present disclosure provides a method for selecting a hairpin oligonucleotide for inhibiting a target protein, said method including the steps of: (a) producing one or more candidate hairpin oligonucleotides comprising a loop region and a double-stranded linear region, wherein the double-stranded linear region comprises a binding or recognition site for the target protein; (b) testing the ability of the one or more candidate hairpin oligonucleotides to bind and/or inhibit the target protein, and (c) selecting a hairpin oligonucleotide which binds and/or inhibits the target protein. Suitably, the hairpin oligonucleotide, inclusive of modifications thereof, and the target protein are that hereinbefore described. In addition to design elements of the present disclosure, there are many known factors to be considered when producing a hairpin oligonucleotide. The specifics depend on the purpose of the oligonucleotide but include features such as strength and stability of the oligonucleotide- target protein interaction, such as the secondary structure of the hairpin oligonucleotide, thermodynamic stability, the position of the DNA-binding domain of the target protein, and/or functional motifs. As used herein, the phrase “inhibits the target protein” or “inhibits an activity of the target protein” or variations thereof means that after administration of a hairpin oligonucleotide to an animal or cell, the animal or cell is not able to elicit a target protein-based biological or cellular
response or is only able to elicit a reduced or partial target protein-based biological or cellular response, such as those biological or cellular responses that are dependent upon the target protein binding to endogenous nucleic acid, when compared to a control or reference sample (e.g., control cells not treated with the candidate oligonucleotide). As such, the present method may include a further step that measures or detects a change in one or more biological activities of the target protein in a cell or an animal in response to the candidate oligonucleotide(s). In some examples, the activity of the target protein is less than about 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2% or 1% of the activity in the absence of the candidate oligonucleotide (e.g., untreated control cells). Accordingly, the level of activity of a target protein may also be compared to a reference or threshold level thereof. Thus, any of the methods disclosed herein may comprise a step of establishing a reference or threshold level activity of the target protein. Once synthesized, candidate hairpin oligonucleotides can be tested for their desired activity using standard procedures in the art. This may involve administering the candidate to cells in vitro expressing the target protein and measuring binding and/or inhibition thereof by the candidate hairpin oligonucleotide (e.g., analysing the amount of transcription of a target gene of the target protein, such as RNA and/or protein) and/or utilising one or more functional assays (e.g., cell growth and proliferation, apoptosis, cellular differentiation) to determine whether the activity thereof changes after exposure to a candidate hairpin oligonucleotide. In another example, the candidate oligonucleotide is administered to an animal, and the animal screened for the amount of target protein inhibition. In other examples, the candidate hairpin oligonucleotide is tested for its ability to bind or hybridize to a target protein, such as by those methods described herein (e.g., assess affinity by EMSA assay). In some examples, inhibitory activity of the candidate hairpin oligonucleotides, and more particularly those containing a binding site for a transcription factor, can be assessed by mRNA reverse transcription quantitative real-time PCR (RT-qPCR) to determine expression of a target gene. For example, RNA can be extracted and purified from cells which have been incubated with a candidate oligonucleotide. cDNA is then synthesized from isolated RNA and RT-qPCR can be performed, using methods and reagents known the art. Protein products and/or downstream cell signalling (and/or one or more markers thereof) of a target gene may alternatively or additionally be determined, such as by any means known in the art, including,
but not limited to, ELISA, western blot, mass spectrometry, proteomics, immunoprecipitation and immunostaining. Suitably, the candidate hairpin oligonucleotide possesses or displays little or no significant off- target and/or nonspecific effects. It is also contemplated that the candidate hairpin oligonucleotide may be rationally designed or engineered de novo based on desired or predicted structural characteristics or features that indicate the candidate oligonucleotide could bind and/or block or inhibit one or more biological activities of the target protein. An initial step of the method may include identifying a plurality of candidate oligonucleotides that are selected according to broad structural and/or functional attributes, such as an ability to bind or specifically bind the target protein. Additionally, the present method may further include one or more of the steps of: selecting the candidate hairpin oligonucleotide that binds (e.g., specifically binds with high affinity) and/or modulates the expression and/or the activity of the target protein; isolating or purifying the candidate hairpin oligonucleotide; formulating the candidate hairpin oligonucleotide into a pharmaceutical formulation; and adding the candidate hairpin oligonucleotide or the pharmaceutical formulation to packaging and/or a container. Accordingly, in another form, the present disclosure provides a hairpin oligonucleotide designed, selected or identified by the present methods. So that preferred embodiments of the present disclosure may be fully understood and put into practical effect, reference is made to the following non-limiting examples. Examples Example 1. This Example tested the ability of the DRpinMyc hairpin oligonucleotide to bind and functionally inhibit the cMyc/Max protein complex.
The Drpin oligonucleotide design described herein differs from antisense oligonucleotides as it is designed to bind proteins and not DNA or RNA. Drpin is a single stranded, self folding, synthetic RNA oligonucleotide, as described in more detail above. The oligonucleotide folds into a hairpin, which usually has a fully Watson and Crick base pairing stem or contains nucleotides that will induce a specific secondary structure, which will be recognised by a target protein (see Figure 1). cMyc was chosen as the target protein to validate this technology with. The Myc family in humans is composed c-myc (MYC), l-myc (MYCL), and n-myc (MYCN). C-MYC is the best characterised as it is involved in up to 70% of human cancers. N-MYC has been shown to be involved in brain cancer, prostate cancers and well a blood cancers. While MYC is one of the common oncogenes driving cancer it has been difficult to target due to is lack of a stable structure. Materials & Methods EMSA assay To confirm the interaction between Drpin and c-Myc and the cMyc:Max heterodimer, electromobility shift assays (EMSA) were performed using the Drpin compound. The oligomeric compounds were labelled with FAM or Cy5, incubated with varying amounts of c- Myc or c-Myc:Max, and bound and free oligomers were separated by native gel electrophoresis. Notably, both c-Myc and c-Myc:Max retarded the migration of the tumour- modulating Drpin with significantly higher affinity than cMyc bound to the same sequence as DNA. Purified cMyc and c-Myc:Max was incubated with 10 nM of labelled oligo for15 min at 37° C in a buffer consisting of 10 mM Tris-HCI (pH8.0), 100 mM NaCl, 0.01 % IGEPAL, 125 mM EDTA and 100 ng/μL BSA. Samples were separated by electrophoresis on a 10% PAGE gel in TBE buffer for 60 min at 80V at 4° C. Molecular dynamics and in silico modelling Drpin Modelling The Drpin oligonucleotide secondary structure was predicted by MC-Fold, accessible as a web server at https://www.major.iric.ca/MC-Fold/. RNA tertiary structure models of Drpin were generated by providing the sequence and MC-Fold secondary structure to RNAcomposer at https://rnacomposer.cs.put.poznan.pl/. Conversion of the RNAComposer tertiary structure to the RP and Sp Phosphorothioate with 2′-O-Methyl chemistry was performed with PyMOL from
Schrödinger, Inc (Schrödinger, L. & DeLano, W., 2020. PyMOL, Available at: http://www.pymol.org/pymol.). Molecular Dynamics Setup and Simulation A novel force field library was required for the ribonucleotide chemistry used in Drpin. The library was created using the R.E.D. server at https://upjv.q4md-forcefieldtools.org/. The Drpin model was solvated in 0.15 M NaCl with TIP3P water molecules added to a padding distance of 15 Å in a truncated octahedron in tleap from the Ambertools16 suite. Atomistic Molecular Dynamics simulations were performed at 300 K for 1 μs for RNA and both stereoisomers of Drpin using pmemd.cuda from the Amber 2020 package. Trajectory Analysis Cpptraj from AmberTools 16 was used to analyse simulation trajectories. K-means Clustering was calculated by sorting into two clusters based on intra-cluster RMSD measured via Davies- Bouldin Index and inter-cluster RMSD measured via pseudo-F. 3D structures of oligomers were processed, and figures were generated with ChimeraX from the University of California San Francisco. Western blotting Western/Immuno blots were performed as described previously (Bolderson et al., 2014, Nucleic Acids Res., 42, 6326–6336) and visualized using an Odyssey infrared imaging system. When necessary, immunoblots were quantified using ImageJ software and normalized to actin. Transfection of DrpinMyc Transfections of Drpin were performed using Lipofectamine RNAimax (Life Technologies) as per manufacturer’s instructions for siRNA transfection. Cell culture Cells were maintained in Roswell Park Memorial Institute medium (DMEM, Sigma). All cell culture media was supplemented with 10% fetal bovine serum (Sigma). Cells were grown in an atmosphere of 21% oxygen and 5% CO2 at 37◦C. Incucyte analysis
A panel of breast cancer cell lines, including MCF7 (hormone positive) and MDA-MB-231 (triple-negative breast cancer) were evaluated to examine the impact of Drpin on cellular proliferation. All cell lines were maintained in DMEM medium supplemented with 10% FBS. All cell lines were cultured at 37°C in a humidified 5% CO2 atmosphere. Lipofectamine RNAimax (Life technologies) was used to aid Drpin delivery. For all cell lines, 2.5 x 103 cells were seeded into clear walled 96-well plates and four regions per well were imaged every two hours over a period of 120 h using the Incucyte Zoom (Essen Biosciences). Image analysis was conducted using the Incucyte Zoom software package. Flow cytometry Cell pellets were created by spinning at 11,000rpm for 3min in eppendorf tubes and removing the supernatant. Pellets were resuspended in 180μl of 70% ethanol and stored at 4°C overnight. The cells were re-pelleted at 14,000g 1min, S/N removed and each sample resuspended in 200μl PI solution (2.2.1) with Rnase A added 1:100v/v (100μg/ml final). Analyse on FACS 2D histogram for PI. Results To determine if the Drpin could bind to cMyc and cMyc:Max, the inventors conducted a EMSA assay with increasing concentrations of Drpin. It was demonstrated that the Drpin binds to cMyc alone and the cMyc:Max dimer with an IC50 of 46 nM and 107 nM respectively (Figure 2). It has been established that the affinity of cMyc:Max for its substrates is typically in excess of 10uM. The inventors next used molecular dynamics and in silico modelling to determine the likely mode of Drpin binding to cMyc:Max. The proposed binding mechanism isillustrated in Figure 3. To explore this further, the inventors next sought to determine if DrpinMyc had an impact on cell growth in cMyc driven tumours. Depletion of cMyc by siRNA does not result in cell death, but does infer a blockade of the cell cycle, and can occur in G1, S or G2 phases of the cell cycle. Triple negative breast cancer is known to be primarily driven by cMyc.
Previous studies have shown that triple negative breast cancer (TNBC) lines have the highest levels of cMyc (reference: (https://www.nature.com/articles/s41419-020-02980-2)). The inventors then sought to treat TNBC cell lines with DrpinMyc. To do this, DrpinMyc was transfected into cells at varying concentrations. As expected, the DrpinMyc oligonucleotide did not kill cells to any great degree over the time period of the experiment (Figure 4). It was confirmed that DrpinMyc was not inducing death under the time points of these experiments. However, it was noticed that treated cells did not appear to be dividing further. To explore this, the inventors next treated cells with DrpinMyc and examined cell doubling/growth using an IncuCyte Zoom® system (Figure 5). A number of concentrations of DrpinMyc were utilised so that an IC50 for growth inhibition could be calculated. The data in Figure 5 generally demonstrates that DrpinMyc causes a dose-dependent reduction in cell proliferation in each the TNBC cell lines. While a loss of growth or proliferation is indicative of cell cycle arrest, this can be confirmed by using flow cytometry using propidium iodide (PI) staining of the DNA. (Figure 6). This demonstrated that TNBC cells were indeed arresting and that cell cycle arrest could result in death over an extended period of time. This phenotype resembles that seen with cells subjected to inhibition of cMyc with siRNA or shRNA. Example 2 Example 1 confirmed that DrpinMyc was capable of arresting cancer cell line growth at a number of different stages of the cell cycle. However, this did not prove on-target activity for this oligonucleotide. To determine this in Example 2, the inventors treated MCF7 and MDA- MB-231 cells with DrpinMyc or with a negative RNA sequence of the same chemistry and investigated gene expression profiles of these respective treatment groups. Materials & methods Cell treatment MCF7 and MDA-MB-231breast cancer cell lines (“NEG”) were treated with vehicle (“MOCK”) or DrpinMyc (“DR”). Transcriptomic differential expression analysis was done for contrasts 231-DR_vs_231-NEG, 231-DR_vs_231-MOCK, MCF7-DR_vs_MCF7-NEG and MCF7-DR_vs_MCF7-MOCK.
RNAseq Each dataset was processed individually for sequencing quality using FastQC. Upon passing quality criteria, reads were aligned against Human genome using STAR aligner. Subsequently, gene and transcript level abundance were estimated with RSEM tool, followed by differential expression analysis using DESeq2 R package. Gene level summarization counts were normalized to library size and batch-effects were corrected for unbiased comparison. Significant differentially expressing genes were identified with significance scores calculated and provided separately for further analysis. Raw data processing & read counting Prior to differential expression (DE) analysis, raw reads were evaluated for quality check in terms of sequencing quality, contamination and over-representation of sequences. Reads were then aligned to latest reference genome (GRCh38) with GTF from Gencode (v39) using STAR aligner (Dobin et al, 2013). Post alignment, efforts have been made to utilise the unmapped reads by trimming to dynamic minimum of 36bp length for realignment. Aligned reads were transformed into read counts per gene/transcript using RSEM tool (Li et al, 2011). Differential expression Pairwise differential expression analysis was performed using DESeq2 R package (Love et al, 2014) with Control/Untreated samples being reference for individual experiments. Briefly, expression counts were scaled and normalized to correct the sequencing depth and batch differences among samples. These normalized counts were then used for differential expression analysis and to generate fold change values in log2 scale for contrasts 231-DR_vs_231-NEG, 231-DR_vs_231-MOCK,MCF7-DR_vs_MCF7-NEGandMCF7-DR_vs_MCF7-MOCK [hereafter fold changes values refer to Log2FC = log2(sample/control)]. Genes with lower read count can generate higher fold change values, which may lead to possible false positives. Hence, to adjust the fold changes that arose due to ratio between lower read counts in samples, the inventors employed a fold change shrinkage estimator approach from DESeq2. Gene prioritization To highlight the statistically significant genes that were differentially expressed, the inventors selected the genes with fold change beyond +1 and adjusted P value lesser than 0.05. Further, for each sample the differential genes were ranked/scored using desiR package (Lazic et al, 2015). Differential genes were assigned score between 0-1, where 1 is highly significant and 0
being failed for the given criteria. Overall significance score was calculated based on a score from fold change, adjusted P value and base mean between contrasts with varying weightage of 100%, 100% and 50% respectively. To illustrate the simple comparison of differentially expressed genes across samples, the overlap of differentially expressed genes with fold change beyond ± 1 and adjusted P values < 0.05 is shown in Figure 7. To elucidate the effect of DrpinMyc treatment, the expression of genes coding for several transcription factors that are critical for regulatory mechanisms were further investigated. In this regard, a subset of the DE gene list was analysed, which was limited to genes that are coding for several TFs retrieved from ChEA database. This data demonstrates differentially expressing TF coding genes separately. The inventors assessed the common and unique differentially expressed transcription factors across all the comparisons (Figures 8 and 9). The ChEA 2016 database (Lachmann A, 2010) was used as a reference for analyzing the transcription factors that overlapped with that of the differential expression data. The ChEA 2016 version consists of 314 transcription factors. The inventors identified the intersecting genes between differentially expressed gene list and ChEA genes (TF coding genes). Results are split for up-and down-regulated genes (Table 1). Results The present Example revealed a number of genes were depleted in the DrpinMyc treated TNBC cells as compared to the mock treated cells (Figures 8 & 9). This RNAseq data further demonstrated that of the top twenty repressed transcripts with DrpinMyc treatment, 11 were known targets of Myc, 4 were known to regulate Myc and one was a target of another E-box transcription factor. The remainder have no known regulator at this time. The main repressed transcription factors were: E2F4, FOXM1, SUZ12 and EZH2. All are direct targets of c-Myc, except E2F4, which functions with Myc to drive certain genes. In conclusion, this data supports an on-target inhibitory effect on cMyc by the inhibitory hairpin oligonucleotide of DrpinMyc. Table 1. Summary statistics of differentially expressed transcription factors
Example 3 Example 1 confirmed that DRpinMyc was capable of inhibiting cancer cell line growth in vitro. This Example tested whether this anti-cancer activity translated to in vivo xenograft cancer models in mice. Materials & Methods Triple-negative breast cancer (TNBC) patient-derived xenograft (PDX) model Seven week old female NOD-scid gamma (NSG) mice were grafted (3rd mammary fat pad) with TNBC PDX tissue. When tumours reached an average size of 50 mm3, animals were treated with DRpin-Myc delivered by tail vein injection (1 injection per week for 6 weeks). Four doses of DRpin-Myc were tested (0, 1, 2.5, 5 mg/kg, 8 animals per dose). Tumour volumes (V=0.5 x length x width2) were normalised to determine the average percent change in tumour volume (tumour volume change (%) = (tumour volume Day x – tumour volume Day 0) / tumour volume Day 0 x 100). TNBC MDA-MB-231 xenograft model Seven week old female NOD-scid gamma (NSG) mice were injected (3rd mammary fat pad) with 2.5 x 106 MDA-MB-231 cells. When tumours reached an average size of 50 mm3, animals were treated with DRpin-Myc delivered by retro-orbital injection (2 injections per week for 4 weeks). Two doses of DRpin-Myc were tested (5 and 10 mg/kg, 6 animals per dose). Tumour volumes (V=0.5 x length x width2) were normalised to determine the average percent change in tumour volume (tumour volume change (%) = (tumour volume Day x – tumour volume Day 0) / tumour volume Day 0 x 100). Results
A dose of 5 mg/kg of DRpin-Myc significantly reduced tumour growth in the TNBC PDX mouse model, without affecting weight gain in treated animals. Doses of 5 and 10 mg/kg of DRpin-Myc also significantly reduced tumour growth in the TNBC MDA-MB-231 xenograft mouse model. Example 4 This Example was performed to develop a model of DRpinMYC and the high-affinity DNA consensus sequence bound to the MYC:MAX complex and provide further data to develop the DRpin platform for controlled selectivity against transcription factors. Methods DRpinMYC model generation. The 2D structure dot-bracket notation was generated through the MC-Fold web server with default settings (Parisien & Major, 2008). The provided DRpinMYC hairpin sequence was 5'- GAGCACGUGGUUAAAAAACCACGUGCUC -3' (SEQ ID NO: 1). This notation was used to create 3D RNA models with RNA Composer (Biesiada, Purzycka, Szachniuk, Blazewicz, & Adamiak, 2016). PS linkages (*) with 2′-OMe (m) were added. RPPS or SPPS stereoisomers were chosen to represent the extreme ranges of structural dynamics imparted by the chirality of PS chemistry. DRpinMYC taking the form, 5′- mG*mA*mG*mC*mA*mC*mG*mU*mG*mG*mU*mU*mA*mA*mA*mA*mA*mA*mC *mC*mA*mC*mG*mU*mG*mC*mU*mC-3′. Conversion of the RNAComposer output to the RPPS and SPPS conformations was carried out using the alter command in PyMOL from Schrödinger, Inc (Schrödinger, L. & DeLano, W., 2020. PyMOL, Available at: http://www.pymol.org/pymol). The model of the high-affinity MYC DNA consensus sequence bound to the MYC:MAX complex was derived by mutating the bases within the duplex of the crystal structure RCSB PDB: 1NKP (Nair & Burley, 2003). Note that the crystal structure contains only the bHLH domains of both proteins. Bound models of DRpinMYC were generated using the MYC: MAX chains of 1NKP. The DNA duplex was removed, and the DRpinMYC models were translated
to reproduce an essential interaction between MYC and the core CG of the E-box sequence 5′- CACGUG-3′ (SEQ ID NO: 12) (Nair & Burley, 2003). Generation of PS and 2′-OMe nucleotide force fields The standard RNA Amber force fields do not include parameters for the PS with 2′-OMe chemistry used herein. Thus, creating a force field library was necessary to represent the modified form of each nucleotide in both 5', 3' and central residues with both RP and SP PS stereoisomers. RESP charges for the library were generated in two runs with RED Server Development (Vanquelef et al., 2011). Partial charges are calculated by constructing the central 2′-OMe nucleotide in the C3′-endo geometry only, as the 2′-OMe is known to enhance this geometry. In calculating the PS charges, dimethyl phosphorothioate in both RP and SP enantiomers was also used in the run. The RESP-A1 method was chosen as this utilises the Hartree-Fock QM method with the 6-31G* basis set for Geometry Optimisation and Molecular Electrostatic Potential calculations in line with the method used to derive the original Amber RNA partial charges (Bayly, Cieplak, Cornell, & Kollman, 1993). Missing P-S bond and O2- P-S and OS-P-S angle parameters were derived from parm99 (Case et al., 2005; Cheatham, Cieplak, & Kollman, 1999). MD system creation Solvent dynamics of oligonucleotide-MYC: MAX systems were studied, and each system was generated with tleap from the AmberTools16 suite (Case et al., 2005). The DNAχOL15 force field was used to represent the DNA systems (Zgarbova et al., 2013; Zgarbova et al., 2011; Zgarbova et al., 2015). MYC and MAX residues were parameterized with the ff14SB protein force field (Maier et al., 2015). Explicit TIP3P water molecules were added to a padding distance of 17 Å in a box, and ionic strength NaCl was added to a concentration of 0.1 M (Mark & Nilsson, 2001). In addition to ionic strength, Na+ ions were added to neutralise the charge. All systems were solvated with ~33,000 water molecules, 100 Na+ and 80 Cl- ions. Oligonucleotide-MYC: MAX MD simulation All simulations were performed in triplicate using pmemd.cuda from the Amber 2020 package (Case et al., 2005; Salomon-Ferrer, Gotz, Poole, Le Grand, & Walker, 2013). Periodic boundary conditions were imposed in all directions. Hydrogen bonds were constrained with the SHAKE method, long-range electrostatic interactions were calculated with the Particle Mesh Ewald technique, and a non-bonded cut-off of 12 Å was used (Essmann et al., 1995;
Ryckaert, Ciccotti, & Berendsen, 1977). The solvent and counterions were minimised using steepest descent for 10,000 steps and conjugate gradient for 5,000 steps. A time step of 2 fs was used throughout equilibration and production. After solvent minimisation, the solvent and counterions were heated from 0 to 300 °K over 50 ns of simulation by applying a 1 kcal/(mol•Å2) harmonic position restraint to the nucleotide heavy atoms with a constant number, volume and temperature (NVT) ensemble. The harmonic restraint was removed, and the system was minimised for 10,000 steps of steepest descent and 5,000 steps of conjugated gradient minimisation. The system was equilibrated by heating from 0 to 300 °K over 50 ns under the prior conditions. Production simulations were run at 300 °K using a Langevin thermostat with a collision frequency of 1.0 ps−1, constant pressure (NPT) at 1 atm, to a total production time frame of 2 μs. Coordinates, velocities, forces and energies were output every 10 ps or 5,000 steps. Trajectory Analysis Cpptraj from AmberTools 16 was used to analyse simulation trajectories (Roe & Cheatham, 2013). The analysis used all 200,000 production frames of each simulation, representing 6 μs of each oligonucleotide. RMSD to the original RNAcomposer models were calculated using all heavy or backbone atoms P, C5', O5', C4', C3', and O3'. K-means Clustering was calculated by sorting into two clusters based on intra-cluster RMSD measured via Davies-Bouldin Index and inter-cluster RMSD measured via pseudo-F. 3D structures were processed, and figures, along with buried surface area calculations, were generated with ChimeraX (Pettersen et al., 2021). MM/GBSA energetics and per residue decomposition. Free energies of binding were calculated with the MM/GBSA method using MMPBSA.py in Amber Tools 16 (Miller et al., 2012). These calculations were run under default parameters using all output trajectory frames from each simulation. The igb method 7 was used on recommendation by the Amber mailing list. idecomp=3 was used to perform pairwise decomposition. These calculations were read and processed using Notepad++ before being converted into heatmaps with the Matplotlib Python library (Hunter, 2007). MM/GBSA, or generalised Born and surface area continuum solvation, is a popular relative free energy of binding technique used to determine the strength by which a ligand binds a receptor. These energetics are typically calculated from molecular dynamics simulations of the
receptor-ligand complex and are more thorough than empirical scoring methods. Ideally, the free energy difference of the binding interaction would be calculated as below (Genheden and Ryde, 2015).
Where [A] represents the ligand in solvent and [B] represents the protein. Unfortunately, the energy contribution to binding would come mostly from solvent-solvent interactions, and the total energy would be far larger than the binding energy. [A]aq + [B] aq ^ [A*B*] aq* (1) In MM/GBSA, free energies are calculated through representative snapshots derived from a given system's molecular dynamics trajectories throughout a simulation. This method uses explicit or implicit solvation simulations with electrostatic contribution calculations to find the solvation free energy, using the Poisson-Boltzmann approach and the same approach to the non-polar solvation free energy to estimate the free energy of binding (Genheden and Ryde, 2015). This thermodynamic cycle is broadly defined by the below equation and visualised in Figure 12: ΔG solv, complex, ΔG solv, receptor, and ΔG solv, ligand is solvation free energies of the ligand-protein complex, the protein, and the ligand, respectively. Free energies are estimated using a continuum Poisson–Boltzmann/surface area approach (Genheden and Ryde, 2015). Results A larger E-box surface area strengthens DRpinMYC binding. From Figure 13, Each simulated system represents 6 μs of simulation time. All systems plateaued at ~3-4 Å within 50 ns; RMSD plots and a clustering summary are provided in Appendix A and B, respectively. The high-affinity MYC consensus sequence demonstrates the more compact arrangement of the B-form DNA geometry. In contrast, the DRpinMYC stereoisomers demonstrate the bulkier arrangement of the A-form geometry imparted by the
2′-OMe chemistry. Both the MYC and MAX basic region and the first helix of the bHLH domain bend to accommodate the backbone interactions of the bulkier DRpinMYC geometry compared to DNA. This bending is made possible by the loop region of the bHLH domain; this loop is largely disordered, allowing the first helix to pivot and impart more interactions between MYC and DRpinMYC than the DNA duplex. The combination of the bulkier A-form major groove of the DRpinMYC stereoisomers and the repositioning of the MYC helix imparts 350- 400 Å2 greater buried surface area between the ligand and complex when compared to DNA. Despite these differences, the interactions between each oligonucleotide and MYC: MAX are highly similar, with many basic amino acid contacts with the oligonucleotide backbone remaining unchanged. Van Der Waals and surface area drive DRpinMYC binding. Table 2. MM/GBSA free energies of binding table
The MM/GBSA derived free binding energy was calculated from the oligonucleotide and MYC: MAX complex simulation trajectories and is provided in Table 2. Both stereoisomers of DRpinMYC demonstrate a clear decrease of -50 kcal/mol in binding energy and, thus, a far stronger interaction of DRpinMYC compared to the high-affinity DNA sequence. The Major driving force behind binding in both cases are electrostatic interactions between backbone PD or PS groups and the basic amino acids of the first helix of MYC and MAX. However, Evdw values demonstrate the greatest change in an individual value of -24 kcal/mol and align with the increased buried surface area in DRpinMYC systems. There is also a notable difference in Esurf, due to a greater surface area with which solvent-solute interaction may occur and is imparted by the bulkier DRpinMYC molecule. Thus, the increased buried surface area seen in Figure 13 imparts stronger or more Van Der Waal interactions between ligand and complex when compared to DNA, while the larger surface area also imparts more solvent-solute interactions.
Despite different geometries, the pairwise interactions of the DNA duplex and the DRpinMYC stereoisomers are highly similar, as seen in Figure 14. There is little change in the MYC or MAX residues involved in binding, and electrostatic interactions between basic amino acids, such as Arginine or Lysine, and the oligonucleotide backbone still dominate. Some base interactions between MYC: MAX and DRpinMYC have shifted by 1-2 bases, and all interactions are more distributed than in the DNA duplex. The 5′ segment of the DRpinMYC hairpin represents the majority of interactions with MYC, while the 3′ segment interacts with MAX. Unlike the DNA duplex, the loop of DRpinMYC and the 5′ bases anterior to the E-box also contribute to the binding. The binding energy decomposition indicates that a similar complex is formed in both DRpinMYC and high-affinity DNA interactions. Discussion For this study, DRpinMYC stereoisomers were manually docked to the MYC: MAX complex, then solvated, simulated for several μs and compared to their DNA counterpart. Systems were analysed by K-means clustering, MM/GBSA and RMSD calculations. The data presented herein indicates that the MYC: MAX complex can bind to an A-form PS-linked hairpin of its high-affinity consensus sequence with a greater affinity than its DNA counterpart. The geometry of the A-form DRpinMYC and the B-form high-affinity DNA duplex differs. MM/GBSA calculations indicate that the MYC: MAX complex binds to DRpinMYC with a stronger affinity than the MYC DNA ligand. Van Der Waals interactions drive this increased affinity due to an increased major groove surface area. Although these interactions are more distributed, as indicated by energy decomposition, the simulation trajectories and clustering indicate that MYC is entirely capable of structural rearrangement to efficiently bind to an A- form geometry. DRpinMYC was partially unwound over the simulation time by its interaction with MYC: MAX. Similar local structural rearrangement is present in the DNA duplex bound to MYC: MAX in the original crystal structure (Nair & Burley, 2003) in which the backbone of the central CG bases of the E-box are pushed towards A-form phi and psi angles. References Adhikary, S., & Eilers, M. (2005). Transcriptional regulation and transformation by Myc proteins. Nat Rev Mol Cell Biol, 6(8), 635-645. doi:10.1038/nrm1703
Bayly, C. I., Cieplak, P., Cornell, W., & Kollman, P. A. (1993). A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. The Journal of Physical Chemistry, 97(40), 10269-10280. doi:10.1021/j100142a004 Biesiada, M., Purzycka, K. J., Szachniuk, M., Blazewicz, J., & Adamiak, R. W. (2016). Automated RNA 3D Structure Prediction with RNAComposer. Methods Mol Biol, 1490, 199- 215. doi:10.1007/978-1-4939-6433-8_13 Cary, P. D., & Kneale, G. G. (2009). Circular dichroism for the analysis of protein-DNA interactions. Methods Mol Biol, 543, 613-624. doi:10.1007/978-1-60327-015-1_36 Case, D. A., Cheatham, T. E., 3rd, Darden, T., Gohlke, H., Luo, R., Merz, K. M., Jr., . . . Woods, R. J. (2005). The Amber biomolecular simulation programs. J Comput Chem, 26(16), 1668-1688. doi:10.1002/jcc.20290 Castell, A., Yan, Q., Fawkner, K., Hydbring, P., Zhang, F., Verschut, V., . .. Larsson, L. G. (2018). A selective high affinity MYC-binding compound inhibits MYC:MAX interaction and MYC-dependent tumor cell proliferation. Sci Rep, 8(1), 10064. doi:10.1038/s41598-018- 28107-4 Cheatham, T. E., 3rd, Cieplak, P., & Kollman, P. A. (1999). A modified version of the Cornell et al. force field with improved sugar pucker phases and helical repeat. J Biomol Struct Dyn, 16(4), 845-862. doi:10.1080/07391102.1999.10508297 Choi, S. H., Mahankali, M., Lee, S. J., Hull, M., Petrassi, H. M., Chatterjee, A. K., ... Shen, W. (2017). Targeted Disruption of Myc-Max Oncoprotein Complex by a Small Molecule. ACS Chem Biol, 12(11), 2715-2719. doi:10.1021/acschembio.7b00799 Chou, K. M., Kukhanova, M., & Cheng, Y. C. (2000). A novel action of human apurinic/apyrimidinic endonuclease: excision of L-configuration deoxyribonucleoside analogs from the 3' termini of DNA. J Biol Chem, 275(40), 31009-31015. doi:10.1074/jbc.M004082200 Crooke, S. T., Seth, P. P., Vickers, T. A., & Liang, X. H. (2020). The Interaction of Phosphorothioate-Containing RNA Targeted Drugs with Proteins Is a Critical Determinant of the Therapeutic Effects of These Agents. J Am Chem Soc, 142(35), 14754-14771. doi:10.1021/jacs.0c04928 Dang, C. V. (2012). MYC on the path to cancer. Cell, 149(1), 22-35. doi:10.1016/j.cell.2012.03.003 Demma, M. J., Mapelli, C., Sun, A., Bodea, S., Ruprecht, B., Javaid, S., ... O'Neil, J. (2019). Omomyc Reveals New Mechanisms To Inhibit the MYC Oncogene. Mol Cell Biol, 39(22). doi:10.1128/MCB.00248-19
Dhanasekaran, R., Deutzmann, A., Mahauad-Fernandez, W. D., Hansen, A. S., Gouw, A. M., & Felsher, D. W. (2022). The MYC oncogene - the grand orchestrator of cancer growth and immune evasion. Nat Rev Clin Oncol, 19(1), 23-36. doi:10.1038/s41571-021-00549-2 Eckstein, F. (2014). Phosphorothioates, essential components of therapeutic oligonucleotides. Nucleic Acid Ther, 24(6), 374-387. doi:10.1089/nat.2014.0506 Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., & Pedersen, L. G. (1995). A Smooth Particle Mesh Ewald Method. Journal of Chemical Physics, 103(19), 8577-8593. doi:Doi 10.1063/1.470117 Fiorentino, F. P., Tokgun, E., Sole-Sanchez, S., Giampaolo, S., Tokgun, O., Jauset, T., . . . Yokota, J. (2016). Growth suppression by MYC inhibition in small cell lung cancer cells with TP53 and RB1 inactivation. Oncotarget, 7(21), 31014-31028. doi:10.18632/oncotarget.8826 Hunter, J. D. (2007). Matplotlib: A 2D Graphics Environment. Computing in Science & Engineering, 9(3), 90-95. doi:10.1109/MCSE.2007.55 Janas, M. M., Jiang, Y., Schlegel, M. K., Waldron, S., Kuchimanchi, S., & Barros, S. A. (2017). Impact of Oligonucleotide Structure, Chemistry, and Delivery Method on In Vitro Cytotoxicity. Nucleic Acid Ther, 27(1), 11-22. doi:10.1089/nat.2016.0639 Llombart, V., & Mansour, M. R. (2022). Therapeutic targeting of "undruggable" MYC. EBioMedicine, 75, 103756. doi:10.1016/j.ebiom.2021.103756 Lourenco, C., Resetca, D., Redel, C., Lin, P., MacDonald, A. S., Ciaccio, R., ... Penn, L. Z. (2021). MYC protein interactors in gene transcription and cancer. Nat Rev Cancer, 21(9), 579- 591. doi:10.1038/s41568-021-00367-9 Maier, J. A., Martinez, C., Kasavajhala, K., Wickstrom, L., Hauser, K. E., & Simmerling, C. (2015). ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J Chem Theory Comput, 11(8), 3696-3713. doi:10.1021/acs.jctc.5b00255 Mark, P., & Nilsson, L. (2001). Structure and Dynamics of the TIP3P, SPC, and SPC/E Water Models at 298 K. The Journal of Physical Chemistry A, 105(43), 9954-9960. doi:10.1021/jp003020w Migawa, M. T., Shen, W., Wan, W. B., Vasquez, G., Oestergaard, M. E., Low, A., ... Seth, P. P. (2019). Site-specific replacement of phosphorothioate with alkyl phosphonate linkages enhances the therapeutic profile of gapmer ASOs by modulating interactions with cellular proteins. Nucleic Acids Res, 47(11), 5465-5479. doi:10.1093/nar/gkz247 Nair, S. K., & Burley, S. K. (2003). X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors. Cell, 112(2), 193-205. doi:10.1016/s0092-8674(02)01284-9
Niu, Z., Liu, H., Zhou, M., Wang, H., Liu, Y., Li, X., ... Li, G. (2015). Knockdown of c-Myc inhibits cell proliferation by negatively regulating the Cdk/Rb/E2F pathway in nasopharyngeal carcinoma cells. Acta Biochim Biophys Sin (Shanghai), 47(3), 183-191. doi:10.1093/abbs/gmu129 Papoulas, O., Williams, N. G., & Kingston, R. E. (1992). DNA binding activities of c-Myc purified from eukaryotic cells. J Biol Chem, 267(15), 10470-10480. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/1587829 Parisien, M., & Major, F. (2008). The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature, 452(7183), 51-55. doi:10.1038/nature06684 Pettersen, E. F., Goddard, T. D., Huang, C. C., Meng, E. C., Couch, G. S., Croll, T. I., . . . Ferrin, T. E. (2021). UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci, 30(1), 70-82. doi:10.1002/pro.3943 Riniker, S. (2018). Fixed-Charge Atomistic Force Fields for Molecular Dynamics Simulations in the Condensed Phase: An Overview. J Chem Inf Model, 58(3), 565-578. doi:10.1021/acs.jcim.8b00042 Roe, D. R., & Cheatham, T. E., 3rd. (2013). PTRAJ and CPPTRAJ: Software for Processing and Analysis of Molecular Dynamics Trajectory Data. J Chem Theory Comput, 9(7), 3084- 3095. doi:10.1021/ct400341p Ryckaert, J.-P., Ciccotti, G., & Berendsen, H. J. C. (1977). Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n-alkanes. Journal of Computational Physics, 23(3), 327-341. doi:https://doi.org/10.1016/0021- 9991(77)90098-5 Sabit, H., Tombuloglu, H., Cevik, E., Abdel-Ghany, S., El-Zawahri, E., El-Sawy, A., ... Al- Suhaimi, E. (2021). Knockdown of c-MYC Controls the Proliferation of Oral Squamous Cell Carcinoma Cells in vitro via Dynamic Regulation of Key Apoptotic Marker Genes. Int J Mol Cell Med, 10(1), 45-55. doi:10.22088/IJMCM.BUMS.10.1.45 Salomon-Ferrer, R., Gotz, A. W., Poole, D., Le Grand, S., & Walker, R. C. (2013). Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs. 2. Explicit Solvent Particle Mesh Ewald. J Chem Theory Comput, 9(9), 3878-3888. doi:10.1021/ct400314y Shen, W., De Hoyos, C. L., Migawa, M. T., Vickers, T. A., Sun, H., Low, A., ... Crooke, S. T. (2019). Chemical modification of PS-ASO therapeutics reduces cellular protein-binding and improves the therapeutic index. Nat Biotechnol, 37(6), 640-650. doi:10.1038/s41587-019-
Vanquelef, E., Simon, S., Marquant, G., Garcia, E., Klimerak, G., Delepine, J. C., . . . Dupradeau, F. Y. (2011). R.E.D. Server: a web service for deriving RESP and ESP charges and building force field libraries for new molecules and molecular fragments. Nucleic Acids Res, 39(Web Server issue), W511-517. doi:10.1093/nar/gkr288 Walhout, A. J., Gubbels, J. M., Bernards, R., van der Vliet, P. C., & Timmers, H. T. (1997). c- Myc/Max heterodimers bind cooperatively to the E-box sequences located in the first intron of the rat ornithine decarboxylase (ODC) gene. Nucleic Acids Res, 25(8), 1493-1501. doi:10.1093/nar/25.8.1493 Wang, Y. H., Liu, S., Zhang, G., Zhou, C. Q., Zhu, H. X., Zhou, X. B., ... Xu, N. Z. (2005). Knockdown of c-Myc expression by RNAi inhibits MCF-7 breast tumor cells growth in vitro and in vivo. Breast Cancer Res, 7(2), R220-228. doi:10.1186/bcr975 Yoo, J., Winogradoff, D., & Aksimentiev, A. (2020). Molecular dynamics simulations of DNA-DNA and DNA-protein interactions. Curr Opin Struct Biol, 64, 88-96. doi:10.1016/j.sbi.2020.06.007 You, S., Lee, H. G., Kim, K., & Yoo, J. (2020). Improved Parameterization of Protein-DNA Interactions for Molecular Dynamics Simulations of PCNA Diffusion on DNA. J Chem Theory Comput, 16(7), 4006-4013. doi:10.1021/acs.jctc.0c00241 Zgarbova, M., Luque, F. J., Sponer, J., Cheatham, T. E., 3rd, Otyepka, M., & Jurecka, P. (2013). Toward Improved Description of DNA Backbone: Revisiting Epsilon and Zeta Torsion Force Field Parameters. J Chem Theory Comput, 9(5), 2339-2354. doi:10.1021/ct400154j Zgarbova, M., Otyepka, M., Sponer, J., Mladek, A., Banas, P., Cheatham, T. E., 3rd, & Jurecka, P. (2011). Refinement of the Cornell et al. Nucleic Acids Force Field Based on Reference Quantum Chemical Calculations of Glycosidic Torsion Profiles. J Chem Theory Comput, 7(9), 2886-2902. doi:10.1021/ct200162x Zgarbova, M., Sponer, J., Otyepka, M., Cheatham, T. E., 3rd, Galindo-Murillo, R., & Jurecka, P. (2015). Refinement of the Sugar-Phosphate Backbone Torsion Beta for AMBER Force Fields Improves the Description of Z- and B-DNA. J Chem Theory Comput, 11(12), 5723- 5736. doi:10.1021/acs.jctc.5b00716
Claims
CLAIMS: 1. A hairpin oligonucleotide for inhibiting a target protein, the hairpin oligonucleotide comprising a loop region and a stem region, wherein the stem region comprises a binding site for the target protein.
2. The hairpin oligonucleotide of Claim 1, wherein the target protein is a transcription factor.
3. The hairpin oligonucleotide of Claim 1 or Claim 2, wherein the transcription factor is an E-box transcription factor.
4. The hairpin oligonucleotide of Claim 3, wherein the transcription factor is selected from the group consisting of Myc, Myc/Max, Fos/Jun, HIF1α/β, MITF, MyoD, HES family, Hey family, ID1/2/3, E2 family, Twist, AHR, AHRR, ARNT, ARNT2, ARNTL, ARNTL2, ASCL1, ASCL2, ASCL3, ASCL4, ATF1, ATF2, ATF4, ATF5, ATF6, ATF7, ATOH1, ATOH7, ATOH8, BACH1, BACH2, BATF, BATF2, BHLHB2, BHLHB3, BHLHB4, BHLHB5, BHLHB8, CLOCK, CREB1, CREB3, CREB3L1, CREB3L2, CREB3L3, CREB3L4, CREB5, CREBL1, CREM, E4BP4, EPAS1, FERD3L, FIGLA, FOSL1, FOSL2, HAND1, HAND2, HES1, HES2, HES3, HES4, HES5, HES6, HES7, HEY1, HEY2, HIF1A, ID1, ID2, ID3, ID4, JUN, JUNB, JUND, KIAA2018, LYL1, MASH1, MATH2, MAX, MESP1, MESP2, MIST1, MITF, MLX, MLXIP, MLXIPL, MNT, MSC, MSGN1, MXD1, MXD3, MXD4, MXI1, MYC, MYCL1, MYCL2, MYCN, MYF5, MYF6, MYOD1, MYOG, NCOA1, NCOA3, NEUROD1, NEUROD2, NEUROD4, NEUROD6, NEUROG1, NEUROG2, NEUROG3, NFE2, NFE2L2, NFE2L3, NHLH1, NHLH2, NPAS1, NPAS2, NPAS3, OAF1, OLIG1, OLIG2, OLIG3, OPAQUE2, PTF1A, SCL, SCXB, SIM1, SIM2, SNFT, SOHLH1, SOHLH2, SREBF1, SREBF2, TAL1, TAL2, TCF12, TCF15, TCF21, TCF3, TCF4, TCFL5, TFAP4, TFE3, TFEB, TFEC, TWIST1, TWIST2, USF1, USF2 and any combination thereof.
5. The hairpin oligonucleotide of Claim 3 or Claim 4, wherein the transcription factor is Myc or Myc/Max.
6. The hairpin oligonucleotide of any one of the preceding claims, wherein the binding site comprises, consists of or consists essentially of the nucleic acid sequence of 5’- CAC[GA]TG-3’, 5’-CAC[GA]UG-3’ or a fragment, variant or derivative thereof.
7. The hairpin oligonucleotide of Claim 6, wherein the binding site of the hairpin oligonucleotide comprises, consists of or consists essentially of the nucleic acid sequence of 5’-GAGCACGUGGUU-3’ (SEQ ID NO: 3) or a fragment, variant or derivative thereof and wherein one or more of the U nucleotides thereof may be a T.
8. The hairpin oligonucleotide of any one of the preceding claims, comprising, consisting of or consisting essentially of the nucleotide sequence of SEQ ID NO: 1 or a fragment, variant or derivative thereof.
9. The hairpin oligonucleotide of any one of the preceding claims, wherein the stem region comprises a sense strand and an antisense strand, each strand having 5 to 50 nucleotides.
10. The hairpin oligonucleotide of any one of the preceding claims, comprising one or a plurality of phosphorothioate internucleotide linkages.
11. The hairpin oligonucleotide of any one of the preceding claims, comprising one or a plurality of modified nucleotides.
12. The hairpin oligonucleotide of Claim 11, wherein the modifications of the modified nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2’-methoxyethyl, 2’-O-alkyl, 2’-O-allyl, 2’-C- allyl, 2’-fluoro, 2’-deoxy, and combinations thereof.
13. The hairpin oligonucleotide of Claim 11 or Claim 12, wherein the modified nucleotides are modified with 2’-OCH3.
14. A pharmaceutical composition comprising the hairpin oligonucleotide of any one of the preceding claims and a pharmaceutically-acceptable carrier, diluent or excipient.
15. A method of inhibiting a target protein of a cell, said method including the step of contacting the cell with the hairpin oligonucleotide of any one of Claims 1 to 13 or the pharmaceutical composition of Claim 14.
16. Use of the hairpin oligonucleotide of any one of Claims 1 to 13 or the pharmaceutical composition of Claim 14 in the manufacture of a medicament for inhibiting a target protein of a cell.
17. A method of preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject, said method including the step of administering to the subject a therapeutically effective amount of the hairpin oligonucleotide of any one of Claims 1 to 13 or the pharmaceutical composition of Claim 14 to thereby prevent, ameliorate or treat the disease, disorder or condition.
18. Use of the hairpin oligonucleotide of any one of Claims 1 to 13 or the pharmaceutical composition of Claim 14 in the manufacture of a medicament for preventing, ameliorating or treating a disease, disorder or condition associated with a target protein in a subject.
19. The method of Claim 17 or the use of Claim 18, wherein the disease, disorder or condition is or comprises a cancer.
20. The method or use of Claim 19, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, cancer of the brain and nervous system, head and neck cancer, colon cancer, colorectal cancer, gastric cancer, liver cancer, kidney cancer, melanoma, skin carcinoma, lymphoid cancer, myelomonocytic cancer, pancreatic cancer, pituitary cancer, bone cancer and soft tissue cancer.
21. The method of use of Claim 19 or Claim 20, wherein the cancer is or comprises breast cancer.
22. A method for selecting a hairpin oligonucleotide for inhibiting a target protein, said method including the steps of: (a) producing one or more candidate hairpin oligonucleotides comprising a loop region and a stem region, wherein the stem region comprises a binding site for the target protein;
(b) testing the ability of the one or more candidate hairpin oligonucleotides to bind and/or inhibit the target protein, and (c) selecting a hairpin oligonucleotide which binds and/or inhibits the target protein.
23. A hairpin oligonucleotide designed, selected or identified by the method of Claim 22.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2022902346 | 2022-08-17 | ||
AU2022902346A AU2022902346A0 (en) | 2022-08-17 | Hairpin Oligonucleotides |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024036377A1 true WO2024036377A1 (en) | 2024-02-22 |
Family
ID=89940231
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/AU2023/050781 WO2024036377A1 (en) | 2022-08-17 | 2023-08-17 | Hairpin oligonucleotides |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024036377A1 (en) |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1357184A2 (en) * | 2002-03-28 | 2003-10-29 | Nanjing Keygen Biotech. Co., Ltd. | Novel cis-element decoys useful as anti-tumor therapeutics |
US20040204374A1 (en) * | 2003-04-11 | 2004-10-14 | Xuegen Wang | Cis-element decoys useful as anti-tumor therapeutics |
US20060074041A1 (en) * | 1997-08-20 | 2006-04-06 | Somagenics, Inc. | Antisense and antigene therapeutics with improved binding properties and methods for their use |
-
2023
- 2023-08-17 WO PCT/AU2023/050781 patent/WO2024036377A1/en unknown
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060074041A1 (en) * | 1997-08-20 | 2006-04-06 | Somagenics, Inc. | Antisense and antigene therapeutics with improved binding properties and methods for their use |
EP1357184A2 (en) * | 2002-03-28 | 2003-10-29 | Nanjing Keygen Biotech. Co., Ltd. | Novel cis-element decoys useful as anti-tumor therapeutics |
US20040204374A1 (en) * | 2003-04-11 | 2004-10-14 | Xuegen Wang | Cis-element decoys useful as anti-tumor therapeutics |
Non-Patent Citations (2)
Title |
---|
HECKER, M ET AL.: "Transcription factor decoy technology: A therapeutic update.", BIOCHEMICAL PHARMACOLOGY, vol. 144, 2017, pages 29 - 34, XP085208276, DOI: 10.1016/j.bcp.2017.06.122 * |
RAD, S ET AL.: "Transcription factor decoy: a pre-transcriptional approach for gene downregulation purpose in cancer.", TUMOR BIOLOGY, vol. 36, 2015, pages 4871 - 4881, XP036224872, DOI: 10.1007/s13277-015-3344-z * |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20220403380A1 (en) | RNA Interactome of Polycomb Repressive Complex 1 (PRC1) | |
JP6738841B2 (en) | Treatment of BDNF-related diseases by inhibition of natural antisense transcripts for brain-derived neurotrophic factor (BDNF) | |
RU2588654C2 (en) | Treatment of sodium channel, voltage-gated, alpha subunit (scna) related diseases by inhibition of natural antisense transcript to scna | |
RU2620980C2 (en) | Treatment of diseases associated with frataxin (fxn), by inhibiting natural antisense fxn transcript | |
RU2585229C2 (en) | Treatment of diseases associated with atonal homolog 1 (aton1) by inhibiting natural antisense transcript of gene aton1 | |
RU2615450C2 (en) | Treating diseases associated with nuclear respiratory factor 1 (nrf1) by inhibition of natural antisense transcript to nrf1 | |
JP6328603B2 (en) | Treatment of TFE3 and insulin receptor substrate 2 (IRS2) related diseases by inhibition of natural antisense transcripts against transcription factor E3 (TFE3) | |
RU2611190C2 (en) | Treatment of diseases related with gene dlg by inhibition of natural antisense transcript of dlg gene | |
ES2762610T3 (en) | Treatment of diseases related to brain-derived neurotrophic factor (BDNF) by inhibition of natural antisense transcript for BDNF | |
ES2656290T3 (en) | Treatment of diseases related to nuclear factor (derived from erythroid 2) similar to 2 (NRF2) by inhibition of natural antisense transcript to NRF2 | |
RU2597972C2 (en) | Treatment of alpha-l-iduronidase (idua) related diseases by inhibition of natural antisense transcript to idua | |
RU2611191C2 (en) | Treatment of diseases, associated with sex hormones binding globulin (shbg), by inhibition of natural antisense transcript to shbg | |
RU2569182C2 (en) | Treating diseases associated with vascular endothelial growth factor (vegf) by suppression of natural antisense vegf transcript | |
ES2760912T3 (en) | Treatment of diseases related to Sirtuin 1 (SIRT1) by inhibition of the natural antisense transcript of Sirtuin 1 | |
JP6025567B2 (en) | Treatment of MBTPS1-related diseases by inhibition of the natural antisense transcript against the membrane-bound transcription factor peptidase, site 1 (MBTPS1) | |
JP5960049B2 (en) | Treatment of antiviral gene-related diseases by suppression of natural antisense transcripts against antiviral genes | |
RU2616283C2 (en) | Treatment of diseases associated with substrate of insulin receptor 2 (irs2) by inhibition of natural antisense transcript to irs2 | |
RU2611187C2 (en) | Treatment diseases, associated with interferon-regulatory factor 8 (irf8), by inhibition of natural antisense transcript to irf8 | |
ES2627763T3 (en) | Treatment of diseases related to the delta 1 homologue (dlk1) by inhibition of natural antisense transcript to dlk1 | |
RU2611195C2 (en) | Treatment of interferon-related developmental regulator 1 (ifrd1) associated diseases by inhibition of natural antisense transcript to ifrd1 | |
ES2658626T3 (en) | Treatment of diseases related to glial cell-derived neurotrophic factor (GDNF) by inhibition of natural antisense transcript to GDNF | |
RU2611192C2 (en) | TREATMENT OF RNase H1 RELATED DISEASES BY INHIBITION OF NATURAL ANTISENSE TRANSCRIPT TO RNase H1 | |
RU2624048C2 (en) | Treatment of diseases associated with the sialidase 4 (neu4) gene, by gene neu4 natural antisense transcript inhibition | |
JP5982362B2 (en) | Treatment of PAR4-related diseases by inhibition of natural antisense transcripts to PAR4 | |
WO2024036377A1 (en) | Hairpin oligonucleotides |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23853759 Country of ref document: EP Kind code of ref document: A1 |