CN115105596A - Tel1在制备降低烟曲霉唑类耐药性的药物中的应用 - Google Patents
Tel1在制备降低烟曲霉唑类耐药性的药物中的应用 Download PDFInfo
- Publication number
- CN115105596A CN115105596A CN202210839637.4A CN202210839637A CN115105596A CN 115105596 A CN115105596 A CN 115105596A CN 202210839637 A CN202210839637 A CN 202210839637A CN 115105596 A CN115105596 A CN 115105596A
- Authority
- CN
- China
- Prior art keywords
- aspergillus fumigatus
- tel1
- drug
- atm
- protein kinase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241001225321 Aspergillus fumigatus Species 0.000 title claims abstract description 68
- 229940091771 aspergillus fumigatus Drugs 0.000 title claims abstract description 66
- 239000003814 drug Substances 0.000 title claims abstract description 56
- 229940079593 drug Drugs 0.000 title claims abstract description 52
- 206010059866 Drug resistance Diseases 0.000 title claims abstract description 20
- 150000003851 azoles Chemical class 0.000 title claims abstract description 14
- 238000002360 preparation method Methods 0.000 title abstract description 10
- SCELLOWTHJGVIC-BGYRXZFFSA-N 2-[(2s,6r)-2,6-dimethylmorpholin-4-yl]-n-[5-(6-morpholin-4-yl-4-oxopyran-2-yl)-9h-thioxanthen-2-yl]acetamide Chemical compound C1[C@@H](C)O[C@@H](C)CN1CC(=O)NC1=CC=C(SC=2C(=CC=CC=2C2)C=3OC(=CC(=O)C=3)N3CCOCC3)C2=C1 SCELLOWTHJGVIC-BGYRXZFFSA-N 0.000 claims abstract description 21
- 101710105755 Serine/threonine-protein kinase tel1 Proteins 0.000 claims abstract description 19
- 102000002804 Ataxia Telangiectasia Mutated Proteins Human genes 0.000 claims abstract description 16
- 239000012827 ATM inhibitor Substances 0.000 claims abstract description 14
- 239000003112 inhibitor Substances 0.000 claims abstract description 10
- 101710178077 Serine/threonine-protein kinase ATM Proteins 0.000 claims abstract description 6
- 239000000725 suspension Substances 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 230000002745 absorbent Effects 0.000 claims description 2
- 239000002250 absorbent Substances 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 239000000853 adhesive Substances 0.000 claims description 2
- 230000001070 adhesive effect Effects 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000003086 colorant Substances 0.000 claims description 2
- 239000003085 diluting agent Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000000839 emulsion Substances 0.000 claims description 2
- 239000008393 encapsulating agent Substances 0.000 claims description 2
- 239000003623 enhancer Substances 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 238000001179 sorption measurement Methods 0.000 claims description 2
- 239000007921 spray Substances 0.000 claims description 2
- 239000004094 surface-active agent Substances 0.000 claims description 2
- 239000000080 wetting agent Substances 0.000 claims description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 abstract description 70
- VHVPQPYKVGDNFY-DFMJLFEVSA-N 2-[(2r)-butan-2-yl]-4-[4-[4-[4-[[(2r,4s)-2-(2,4-dichlorophenyl)-2-(1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazin-1-yl]phenyl]-1,2,4-triazol-3-one Chemical compound O=C1N([C@H](C)CC)N=CN1C1=CC=C(N2CCN(CC2)C=2C=CC(OC[C@@H]3O[C@](CN4N=CN=C4)(OC3)C=3C(=CC(Cl)=CC=3)Cl)=CC=2)C=C1 VHVPQPYKVGDNFY-DFMJLFEVSA-N 0.000 abstract description 25
- 229960004130 itraconazole Drugs 0.000 abstract description 25
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 230000002401 inhibitory effect Effects 0.000 abstract description 7
- 201000002909 Aspergillosis Diseases 0.000 abstract description 6
- 208000036641 Aspergillus infections Diseases 0.000 abstract description 6
- 239000003596 drug target Substances 0.000 abstract description 2
- 238000012216 screening Methods 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 11
- 108010004586 Ataxia Telangiectasia Mutated Proteins Proteins 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 9
- 206010028980 Neoplasm Diseases 0.000 description 8
- 241000233866 Fungi Species 0.000 description 7
- 230000009471 action Effects 0.000 description 7
- 229940121375 antifungal agent Drugs 0.000 description 7
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 7
- 230000012010 growth Effects 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 235000018102 proteins Nutrition 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- BCEHBSKCWLPMDN-MGPLVRAMSA-N voriconazole Chemical compound C1([C@H](C)[C@](O)(CN2N=CN=C2)C=2C(=CC(F)=CC=2)F)=NC=NC=C1F BCEHBSKCWLPMDN-MGPLVRAMSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 229960004740 voriconazole Drugs 0.000 description 6
- 206010021143 Hypoxia Diseases 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 210000003470 mitochondria Anatomy 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 230000005778 DNA damage Effects 0.000 description 4
- 231100000277 DNA damage Toxicity 0.000 description 4
- 230000000843 anti-fungal effect Effects 0.000 description 4
- 239000003429 antifungal agent Substances 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 230000002438 mitochondrial effect Effects 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 230000036542 oxidative stress Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000004885 tandem mass spectrometry Methods 0.000 description 4
- 230000003827 upregulation Effects 0.000 description 4
- 206010003594 Ataxia telangiectasia Diseases 0.000 description 3
- 101150033031 CYP51A gene Proteins 0.000 description 3
- 108010077544 Chromatin Proteins 0.000 description 3
- 230000005971 DNA damage repair Effects 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 3
- 102000004142 Trypsin Human genes 0.000 description 3
- 108090000631 Trypsin Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 210000003483 chromatin Anatomy 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 230000007954 hypoxia Effects 0.000 description 3
- 201000009085 invasive aspergillosis Diseases 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 239000001965 potato dextrose agar Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000012588 trypsin Substances 0.000 description 3
- XRKYMMUGXMWDAO-UHFFFAOYSA-N 2-(4-morpholinyl)-6-(1-thianthrenyl)-4-pyranone Chemical class O1C(C=2C=3SC4=CC=CC=C4SC=3C=CC=2)=CC(=O)C=C1N1CCOCC1 XRKYMMUGXMWDAO-UHFFFAOYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010006473 Bronchopulmonary aspergillosis Diseases 0.000 description 2
- 101000777277 Homo sapiens Serine/threonine-protein kinase Chk2 Proteins 0.000 description 2
- 102100021695 Lanosterol 14-alpha demethylase Human genes 0.000 description 2
- 101710146773 Lanosterol 14-alpha demethylase Proteins 0.000 description 2
- 108090000430 Phosphatidylinositol 3-kinases Proteins 0.000 description 2
- 102000001253 Protein Kinase Human genes 0.000 description 2
- 208000004430 Pulmonary Aspergillosis Diseases 0.000 description 2
- PLXBWHJQWKZRKG-UHFFFAOYSA-N Resazurin Chemical compound C1=CC(=O)C=C2OC3=CC(O)=CC=C3[N+]([O-])=C21 PLXBWHJQWKZRKG-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- 102100031075 Serine/threonine-protein kinase Chk2 Human genes 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 208000006778 allergic bronchopulmonary aspergillosis Diseases 0.000 description 2
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 2
- 229960003942 amphotericin b Drugs 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000008686 ergosterol biosynthesis Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 208000005017 glioblastoma Diseases 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000004898 mitochondrial function Effects 0.000 description 2
- 238000003068 pathway analysis Methods 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- RAGOYPUPXAKGKH-XAKZXMRKSA-N posaconazole Chemical compound O=C1N([C@H]([C@H](C)O)CC)N=CN1C1=CC=C(N2CCN(CC2)C=2C=CC(OC[C@H]3C[C@@](CN4N=CN=C4)(OC3)C=3C(=CC(F)=CC=3)F)=CC=2)C=C1 RAGOYPUPXAKGKH-XAKZXMRKSA-N 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 108060006633 protein kinase Proteins 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 230000008261 resistance mechanism Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- HWCKGOZZJDHMNC-UHFFFAOYSA-M tetraethylammonium bromide Chemical compound [Br-].CC[N+](CC)(CC)CC HWCKGOZZJDHMNC-UHFFFAOYSA-M 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- SCELLOWTHJGVIC-WOJBJXKFSA-N 2-[(2r,6r)-2,6-dimethylmorpholin-4-yl]-n-[5-(6-morpholin-4-yl-4-oxopyran-2-yl)-9h-thioxanthen-2-yl]acetamide Chemical compound C1[C@@H](C)O[C@H](C)CN1CC(=O)NC1=CC=C(SC=2C(=CC=CC=2C2)C=3OC(=CC(=O)C=3)N3CCOCC3)C2=C1 SCELLOWTHJGVIC-WOJBJXKFSA-N 0.000 description 1
- LHGMHYDJNXEEFG-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]iminocyclohexa-2,5-dien-1-one Chemical compound C1=CC(N(C)C)=CC=C1N=C1C=CC(=O)C=C1 LHGMHYDJNXEEFG-UHFFFAOYSA-N 0.000 description 1
- 229940122485 ATM kinase inhibitor Drugs 0.000 description 1
- 206010000830 Acute leukaemia Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 101150065175 Atm gene Proteins 0.000 description 1
- 102100025048 Cell cycle checkpoint control protein RAD9A Human genes 0.000 description 1
- ACTIUHUUMQJHFO-UHFFFAOYSA-N Coenzym Q10 Natural products COC1=C(OC)C(=O)C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UHFFFAOYSA-N 0.000 description 1
- 230000009946 DNA mutation Effects 0.000 description 1
- 102000008013 Electron Transport Complex I Human genes 0.000 description 1
- 108010089760 Electron Transport Complex I Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 201000010915 Glioblastoma multiforme Diseases 0.000 description 1
- 101100057233 Homo sapiens ATM gene Proteins 0.000 description 1
- 101001077508 Homo sapiens Cell cycle checkpoint control protein RAD9A Proteins 0.000 description 1
- 101000615488 Homo sapiens Methyl-CpG-binding domain protein 2 Proteins 0.000 description 1
- 101001128138 Homo sapiens NACHT, LRR and PYD domains-containing protein 2 Proteins 0.000 description 1
- 101000981336 Homo sapiens Nibrin Proteins 0.000 description 1
- 101000785063 Homo sapiens Serine-protein kinase ATM Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 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
- 102220595357 Lanosterol 14-alpha demethylase_L98H_mutation Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102100021299 Methyl-CpG-binding domain protein 2 Human genes 0.000 description 1
- 102100024403 Nibrin Human genes 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000038030 PI3Ks Human genes 0.000 description 1
- 108091007960 PI3Ks Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 102000003993 Phosphatidylinositol 3-kinases Human genes 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108010031271 Saccharomyces cerevisiae Proteins Proteins 0.000 description 1
- 101710151717 Stress-related protein Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000009098 adjuvant therapy Methods 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 239000011717 all-trans-retinol Substances 0.000 description 1
- 239000012080 ambient air Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000032770 biofilm formation Effects 0.000 description 1
- 238000003766 bioinformatics method Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000025084 cell cycle arrest Effects 0.000 description 1
- 230000006369 cell cycle progression Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 1
- 235000017471 coenzyme Q10 Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000010485 coping Effects 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
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000035784 germination Effects 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 238000011134 hematopoietic stem cell transplantation Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 102000043380 human ATM Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 1
- VHVPQPYKVGDNFY-ZPGVKDDISA-N itraconazole Chemical compound O=C1N(C(C)CC)N=CN1C1=CC=C(N2CCN(CC2)C=2C=CC(OC[C@@H]3O[C@](CN4N=CN=C4)(OC3)C=3C(=CC(Cl)=CC=3)Cl)=CC=2)C=C1 VHVPQPYKVGDNFY-ZPGVKDDISA-N 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004879 molecular function Effects 0.000 description 1
- 101150071637 mre11 gene Proteins 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 238000009520 phase I clinical trial Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 229960001589 posaconazole Drugs 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 238000000575 proteomic method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 101150010682 rad50 gene Proteins 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- NPCOQXAVBJJZBQ-UHFFFAOYSA-N reduced coenzyme Q9 Natural products COC1=C(O)C(C)=C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)C(O)=C1OC NPCOQXAVBJJZBQ-UHFFFAOYSA-N 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 230000009131 signaling function Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000007474 system interaction Effects 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 229940035936 ubiquinone Drugs 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- -1 voriconazole) Chemical class 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 101150020580 yap1 gene Proteins 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
-
- 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
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本发明公开了一种Tel1在制备降低烟曲霉唑类耐药性的药物中的应用，具体为丝氨酸/苏氨酸蛋白激酶Tel1在制备降低烟曲霉唑类耐药性的药物中的应用，该药物优选为Tel1/ATM抑制剂KU‑60019。本发明首次发现了丝氨酸/苏氨酸蛋白激酶Tel1与烟曲霉唑类耐药性存在特殊相关性，作为烟曲霉唑类耐药性的药物靶标，可应用于避免或者降低烟曲霉唑类耐药株耐药性的曲霉病治疗药物的制备和筛选；此外，本发明进一步提供了一种抑制丝氨酸/苏氨酸蛋白激酶Tel1/ATM的抑制剂KU‑60019，可明显增加烟曲霉对曲霉病药物伊曲康唑的敏感性。
Description
技术领域
本发明属于生物医药领域，尤其涉及一种丝氨酸/苏氨酸蛋白激酶Tel1在制备降低烟曲霉唑类耐药性的药物中的应用。
背景技术
烟曲霉(Aspergillusfumigatus)是最常见的曲霉属真菌，其分生孢子是环境空气标本中真菌的主要形态。人体从气道吸入的孢子若不能排出，可引起侵袭性曲霉病、慢性肺曲霉病、过敏性曲霉病和脑曲霉病等。由于细胞毒性药物、糖皮质激素、靶向药物、新冠肺炎流行、造血干细胞移植和器官移植等，侵袭性曲霉病的发病率逐年升高。在严重免疫功能低下的患者中，曲霉病已成为最常见的死亡原因之一，急性白血病和造血干细胞移植者患曲霉病的病死率高达40％～50％。
侵袭性曲霉病首选伏立康唑治疗，慢性肺曲霉病和过敏性曲霉病多用伊曲康唑和伏立康唑治疗。但是，近年来全世界烟曲霉对唑类抗真菌药物的敏感性显著下降。例如2004年英国曼彻斯特报道烟曲霉临床分离株唑类耐药率5％，2009年已达20％。目前中国和全球最常见的烟曲霉唑类耐药基因型为TR34/L98H突变型。
唑类抗真菌药与14-α-甾醇脱甲基酶(CYP51)结合，抑制烟曲霉细胞膜麦角甾醇的生物合成。cyp51A突变是烟曲霉临床株唑类耐药最常见的原因，我们与其他研究者发现cyp51A突变是通过修饰结合位点来影响唑类亲和力。非cyp51A突变所致唑类耐药的机制包括：(1)外排泵过度表达：AfuMdr4与伏立康唑耐药相关，Cdr1B与伊曲康唑耐药相关；(2)CYP51过度表达：SrbA、HapE和AtrR缺失可导致唑类耐药；(3)应对氧化应激：烟曲霉Yap1突变导致伏立康唑耐药；(4)线粒体参与：有证据表明线粒体复合体I可以消除唑类导致的活性氧释放对烟曲霉的毒性，从而提高对唑类药物的耐受。
生物膜是附着于物体表面，被胞外基质包裹的有组织的微生物群体，具有与游离状态时不同的组织成分。尽管以往的科学研究多以微生物在液态培养基中浮游生长或者固态培养基中菌落生长为基础，现在公认对于绝大多数微生物来说生物膜附着更能反映其自然生长状态。烟曲霉生物膜主要由菌丝骨架和胞外基质组成，形成过程可分为三个阶段：分生孢子萌发和粘附、孢子生成菌丝(10～12小时)、生物膜形成及成熟(48～72小时)。生物膜可以帮助真菌抵抗药物的作用，生物膜导致的耐药机制有：细胞外基质屏障、药物外排泵的过度表达、缺氧耐受、菌体细胞休眠和生物膜-宿主免疫系统相互作用等。
发明内容
为克服现有技术的缺点和不足，本发明的目的在于提供一种丝氨酸/苏氨酸蛋白激酶Tel1的抑制剂用于增强烟曲霉对唑类药物的敏感性，即做类药物的增效剂。
本发明是这样实现的，丝氨酸/苏氨酸蛋白激酶Tel1在制备降低烟曲霉唑类耐药性的药物中的应用。
优选地，所述药物为有效剂量的抑制丝氨酸/苏氨酸蛋白激酶Tel1/ATM的抑制剂。
优选地，所述药物为Tel1/ATM抑制剂KU-60019。
优选地，所述药物包括药物学上可以接受的载体；所述载体为稀释剂、缓冲剂、混悬剂、乳剂、颗粒剂、包囊剂、赋形剂、填充剂、粘合剂、喷雾剂、透皮吸收剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、着色剂、矫味剂或吸附载体。
本发明克服现有技术的不足，公开了Tel1在制备降低烟曲霉唑类耐药性的药物中的应用。丝氨酸/苏氨酸蛋白激酶Tel1在哺乳动物直系同源物(homolog ous protein)是共济失调毛细血管扩张突变蛋白激酶(ataxia-telangiectasia mutated protein kinases，ATM蛋白激酶)。丝氨酸/苏氨酸蛋白激酶Tel1/ATM属于磷脂酰肌醇-3-激酶相关蛋白激酶(phosphatidylinositol3-kinase-related kinase，PIKK)家族。ATM基因于1995年在共济失调毛细血管扩张症(Ataxia Telangiectasia)的研究中首次得到克隆(A single ataxiatelangiectasia gene witha product similar to PI-3kinase,Science,268(1995)1749-1753.)。2000年Tel1在酿酒酵母(Saccharomyces cerevisiae)中被发现(J.C.Mallory,T.D.Petes,Protein kinase activity of Tel1p and Mec1p,twoSaccharomyces cerevisiaeproteins related to the human ATM protein kinase,ProcNatl Acad Sci U SA,97(2000)13749-13754.)。真菌Tel1与人类ATM基因高度同源，基因产物蛋白结构也高度保守和相似。
如图1所示，Tel1/ATM具有以下重要功能：(1)DNA损伤修复：当DNA损伤发生时，ATM迅速被激活，与DNA损伤部位的MRN(Mre11/Rad50/NBS1)复合物共信号传导，还激活了数百个传感器和效应器的DNA损伤修复信号通路的级联，从而启动DNA损伤修复反应；(2)细胞周期停滞：直接磷酸化并稳定p53，诱导细胞G1/S和G2/M周期停滞，防止未修复的DNA突变传递到子代细胞中；(3)凋亡：当DNA损伤太严重而无法有效修复时，激活的p53可诱导细胞凋亡；(4)细胞周期检查：激活细胞周期调控蛋白RAD9A、检查点激酶(checkpoint kinase，CHK)1和CHK2等使细胞周期进展暂时停止，以实现DNA损伤修复；(5)染色质改变修复：将染色质改变信号传导到CHK2和p53等底物，继而活化多个通路以维持染色质完整性；(6)氧化压力应激；(7)抑制线粒体功能；和(8)缺氧应激。
ATM抑制剂可抑制多种肿瘤耐药，因此，若干ATM抑制剂被开发和用于肿瘤辅助治疗。比如，多形性胶质母细胞瘤(Glioblastoma multiform)是最常见的脑实质肿瘤，患者通常在一年内死于该疾病。ATM抑制剂KU-60019与放射治疗联合使用时，可显著诱导该肿瘤消退和提高动物存活率，此抑制剂已进入I期临床试验。
为了验证Tel1/ATM抑制剂KU-60019是否通过抑制真菌的Tel1从而增强唑类药物抑菌效果，本发明将Tel1/ATM抑制剂KU-60019与烟曲霉和伊曲康唑共孵育，进行药敏实验。结果发现在抑制剂KU-60019作用下，伊曲康唑对烟曲霉标准株的最小抑菌浓度值(MIC值)降低为无抑制剂时的1/4，耐药株伊曲康唑MIC值降低到无抑制剂的1/2～1/8。实验结果证明，Tel1/ATM抑制剂KU-60019可明显增加烟曲霉对伊曲康唑敏感性，并且印证Tel1参与了烟曲霉唑类耐药。
唑类药物可以导致烟曲霉线粒体活性氧泄露，靶向烟曲霉细胞膜，从而破坏氧化还原稳态。但是烟曲霉可通过抑制线粒体呼吸复合物I(Mitochondrial Complex I，NADH:ubiquinone oxidoreductase，泛醌氧化还原酶)，消除升高的活性氧。烟曲霉唑类耐药与线粒体复合物I相关，线粒体复合物I能力丧失或抑制会导致烟曲霉对唑类耐药。Tel1/ATM激活可抑制细胞线粒体功能，应对氧化应激。这也是人肿瘤ATM上调，导致耐药的机制之一。
生物膜也是烟曲霉应对唑类的重要方法。烟曲霉生物膜在其成熟过程中，始终保持缺氧微环境的空间梯度。即使丝状真菌生物膜内菌丝之间有充足的氧气，线粒体耗氧还是会自我诱导出缺氧区。由于缺氧等原因，生物膜基部菌丝代谢状态降低。然而唑类(特别是伏立康唑)需要代谢活性才能有效地影响麦角甾醇合成，发挥抗真菌活。这些缺氧区的形成，不仅有助于烟曲霉生物膜生长，还有助于增加抗真菌药物耐药性。而Tel1/ATM激活有助于细胞应对缺氧，有助于真菌在生物膜中生长。Tel1/ATM应对缺氧在肿瘤耐药机制中发挥一定作用。
综合分析Tel1/ATM功能、人肿瘤ATM上调导致耐药以及烟曲霉唑类耐药的机制后，判定烟曲霉唑类耐药株生物膜蛋白组学分析中Tel1显著上调，是为了更好地应对唑类药物压力。具体表现为：
(1)烟曲霉唑类耐药株Tel1上调可抑制线粒体功能和降低活性氧水平
唑类药物引起烟曲霉内源性活性氧增多，烟曲霉唑类耐药株在唑类压力下Tel1表达明显上调，从而抑制线粒体和减少释放的活性氧，导致其生物膜抗药性进一步增强。在本发明的蛋白组学结果中，耐药株在伊曲康唑压力下，氧化应激相关蛋白过氧化物酶Asp f3表达也都明显上调，也支持了本发明的假设。
(2)烟曲霉唑类耐药株Tel1上调有助于适应生物膜缺氧微环境
烟曲霉生物膜在其成熟过程中会形成缺氧微环境，Tel1通路激活有助于克服生物膜基底层菌丝的缺氧问题，从而更好地应对缺氧并发展耐药倾向。
相比于现有技术的缺点和不足，本发明具有以下有益效果：
(1)本发明首次发现了丝氨酸/苏氨酸蛋白激酶Tel1与烟曲霉唑类耐药性存在特殊相关性，作为烟曲霉唑类耐药性的药物靶标，可应用于避免或者降低烟曲霉唑类耐药株耐药性的曲霉病治疗药物的制备和筛选；
(2)本发明进一步提供了一种抑制丝氨酸/苏氨酸蛋白激酶Tel1/ATM的抑制剂KU-60019，可明显增加烟曲霉对曲霉病药物伊曲康唑的敏感性。
附图说明
图1是是丝氨酸/苏氨酸蛋白激酶Tel1/ATM的主要信号通路和功能示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
1、伊曲康唑作用下烟曲霉生物膜中丝氨酸/苏氨酸蛋白激酶Tel1的变化
1.1、抗真菌剂、菌株准备
本发明实施例中烟曲霉菌株和抗真菌剂的制备涉及两株烟曲霉(菌种来源详见：Liu M,Zeng R,Zhang L,Li D,Lv G,Shen Y,Zheng H,Zhang Q,Zhao J,Zheng N,LiuW.Multiple cyp51A-based mechanisms identified in azole-resistant isolatesofAspergillus fumigatus from China.Antimicrob Agents Chemother.2015Jul；59(7):4321-5.doi:10.1128/AAC.00003-15.Epub 2015Apr 20.PMID:25896700；PMCID:PMC4468671.)，包括Af293(野生型菌株)和Shjt42b(具有TR34/L98H基因型cyp51A的唑类临床分离株)。A.fumigatus菌株在马铃薯葡萄糖琼脂(PDA)上维持在4℃，随后在PDA斜坡培养基上以37℃培养72h。对于抗真菌剂，伊曲康唑以粉末形式购自美国密苏里州圣路易斯的Sigma-Aldrich。
1.2、生物膜生长
生物膜生长按照Reichhardt,C等人的描述进行，稍作修改(参考文献：C.Reichhardt,J.A.Ferreira,L.M.Joubert,K.V.Clemons,D.A.Stevens,L.Cegelski,Analysis ofthe Aspergillus fumigatus Biofilm ExtracellularMatrix by Solid-State NuclearMagnetic Resonance Spectroscopy,Eukaryot Cell,14(2015)1064-1072.)。现有研究表明，烟曲霉的生物膜活力发生明显变化，当Af293在0.25μg/mL浓度的伊曲康唑下生长，而Shjt42b在256μg/mL浓度的伊曲康唑下生长(参考文献：M.Liu,H.Zheng,R.Zeng,G.Liang,N.Zheng,W.Liu,Effects of Itraconazole and Micafungin onAspergillus fumigatus Biofilms,Mycopathologia,(2021))。
将在100mL RPMI 1640培养基中含有1×105分生孢子/mL的培养物悬浮液接种在每个500mL聚苯乙烯组织培养瓶中；将烧瓶在37℃下静置培养96小时，加入或不加入0.25～256mg/mL的伊曲康唑；收获的生物膜用100mL磷酸盐缓冲盐水(PBS)原位洗涤两次，以去除浮游细胞和松散粘附的细胞；在每个烧瓶中用40mL 10mMTris(pH 7.4)淹没固着细胞，并用涡旋混合器温和混合60秒，添加蛋白酶抑制剂(体积比为1000:1)(Solarbio，A8260)；将每个样品转移至50mL锥形管中，并在4℃下储存直至处理。
1.3、蛋白质提取和消化
(1)对于ECM提取，将含有烟曲霉生物膜的管进行超声处理10分钟，然后在涡旋混合器上高速混合30秒。
(2)将试管在4～C下以3,600g离心15分钟。将上清液倒入新试管中并再次离心以除去额外的细胞。将上清液转移至新的超滤离心管(Merck Millipore；Amicon Ultra-50，Ultracel-3k)，4℃，5000g 90min，离心浓缩回收至新离心管中，使用BCA试剂盒测定蛋白浓度，将上清液在液氮中快速冷冻并冻干。
为了消化，蛋白质溶液在56℃下用5mM二硫苏糖醇还原30分钟，然后在室温下在黑暗中用11mM碘乙酰胺烷基化15分钟。然后通过添加100mM TEAB到尿素浓度小于2M来稀释蛋白质样品。最后在1处加入胰蛋白酶：第一次消化过夜时胰蛋白酶与蛋白质的质量比为50，第二次消化4小时时胰蛋白酶与蛋白质的质量比为1：100。
1.4、TMT标记和HPLC分级
将胰蛋白酶肽用0.5M TEAB溶解并根据TMT试剂盒操作说明进行标记。标记后，用8％至32％乙腈(pH 9.0)梯度在60分钟内将肽分离成60个级分。
将它们合并成18个级分并通过真空离心干燥。使用了十二种培养物(两种菌株的每种处理和未处理样品的三个生物学重复)。
1.5、LC-MS/MS分析
将多肽溶解在0.1％甲酸(溶剂A)中，直接上样到自制的反相分析柱(15cm长，75μmi.d.)上。梯度包括从6％增加到22％的溶剂B(0.1％甲酸在98％乙腈中)42分钟内，22％到30％在2分钟内并在3分钟内上升到80％，然后在80％下保持至少3分钟，所有这些都在500nL/min的恒定流速下在EASY-nLC1000UPLC系统上。
对肽进行NSI源处理，然后在与UPLC在线耦合的Q ExactiveTM Plus(Thermo)中进行串联质谱(MS/MS)。施加的电喷雾电压为2.0kV，全扫描的m/z扫描范围为350至1550，在Orbitrap中以60,000的分辨率检测到完整的肽段，然后选择肽用于MS/MS，并在Orbitrap中以15,000的分辨率检测到片段。在一次MS扫描和20次MS/MS扫描之间交替进行，动态排除时间为15.0秒。自动增益控制(AGC)设置为5E4，固定的第一质量设置为100m/z。
1.6、数据库研究
使用Maxquant搜索引擎(v.1.5.2.8)处理生成的MS/MS数据。该数据库是烟曲霉(9647个序列)。添加逆库以计算由于随机匹配导致的误报率(FDR)。数据库中增加了常见污染库，以消除污染蛋白质对鉴定结果的影响。酶切设定为胰蛋白酶/P，错过的切割位点数设置为2，肽的最小长度设置为7，肽修饰的最大数量设置为5。主要母离子的质量误差容限设置为20ppm和5ppm分别用于First search和Main search。二级碎片离子的质量误差容限设置为0.02Da。半胱氨酸烷基化设置为固定修饰，可变修饰是蛋氨酸的氧化，蛋白质N-末端的乙酰化，脱酰胺(NQ)。定量方法设置为TMT-6plex，蛋白质鉴定和PSM鉴定的FDR设置为1％。
1.7、生物信息学分析
根据检索结果和质谱分析，本发明使用Proteome Discoverer软件进行定量蛋白质组学分析。方差分析(ANOVA)用于评估统计显着性。
P值<0.05和倍数变化(FC)≥2或FC≤0.5被认为表示显着变化。通过以下方法进行功能富集分析以确定差异表达蛋白(DEPs)的作用：
(1)使用DAVID数据库(https://david.ncifcrf.gov/)基于三类(生物过程、细胞成分和分子功能鉴定基因本体)进行(GO)富集分析。
(2)使用KEGG数据库(http://www.genome.jp/kegg/pathway.html)进行京都基因和基因组百科全书(KEGG)通路分析，使用FungiFun 2.2.8BETA软件(https://elbe.hki-jena.de/fungifun/fungifun.php)对DEP进行细胞通路分析。
结果如下表1所示：
表1真菌生物膜Tel1在唑类药物作用下的检测结果
表1中，Shjt42b_ITC256/Shjt42b_ITC0 Ratio：耐药株shjt42b在有伊曲康唑(256μg/mL)比无伊曲康唑的比值；Shjt42b_ITC256/Af293_ITC0.25 Ratio：耐药株shjt42b在伊曲康唑作用下(256μg/mL)比上标准株af293在伊曲康唑0.25μg/mL作用下的比值。
由上表1可知，本发明通过对烟曲霉生物膜基质(ExtracellularMatrix)成分进行了蛋白质组学分析后发现，烟曲霉生物膜中丝氨酸/苏氨酸蛋白激酶Tel1在伊曲康唑作用下会显著升高，本发明在使用唑类药物处理烟曲霉的时候，发现它们的生物膜中的丝氨酸/苏氨酸蛋白激酶Tel1会显著升高。
2、ATM抑制剂KU-60019的药敏实验
ATM抑制剂(KU-60019，参考文献：Golding SE,Rosenberg E,Valerie N,etal.Improved ATM kinase inhibitor KU-60019radiosensitizes glioma cells,compromises insulin,AKT and ERK prosurvival signaling,and inhibits migrationand invasion.Mol Cancer Ther.2009；8(10):2894-2902.)是一种改进的KU-55933类似物，在无细胞试验中作用于ATM，IC50为6.3nM，作用于ATM比作用于DNA-PK和ATR的选择性分别高270和1600倍，并且是高度有效的放射增敏剂。
KU-60019是一种改进的KU-55933类似物，分子式：C30H32N3O5S，化学名称：(2R,6S)-2,6-二甲基-N-[5-[6-(4-吗啉基)-4-氧代-4H-吡喃-2-基]-9H-噻吨-2-基]-4-吗啉乙酰胺。KU-60019、KU-55933的结构式分别为：
将ATM抑制剂溶于水，终浓度为3μM；储存条件：-20℃，粉末，3年，或者，-80℃，溶于溶剂，2年。
使用方法：使用KU-60019处理细胞1、3和5天。通过AlamarBlue测定细胞生长：将Alamar Blue(10％溶液)加到培养基中，达到终浓度1％，实验板在37℃下温育1小时，在Fluoro-Count酶标仪上(激发光530nm，发射光590nm)测定荧光值，测定结果作为衡量细胞生长的一个指标，通过台酚蓝/荧光激活的细胞分选(FACS)实验测定细胞存活。
3、Tel1与烟曲霉唑类耐药的关系
为了验证Tel1是否参与烟曲霉唑类耐药，本发明将Tel1/ATM抑制剂KU-60019与烟曲霉和伊曲康唑共孵育，进行药敏实验。实验过程参照美国临床实验室标准化研究所CLSIM-38A2推荐的丝状真菌药敏指南，使用微量液基稀释法并稍作改进，将唑类抗真菌药物进行梯度稀释后与烟曲霉菌悬液加入96孔细胞培养板中，再加入KU-60019，37℃培养46～50h后读数，肉眼观察到无菌生长的最低药物浓度即为MIC。如下表2所示：
表2有/无Tel1抑制剂作用下的烟曲霉伊曲康唑的MIC值
由表2可知，KU-60019终浓度为5mM时，标准株(AF293)伊曲康唑MIC值降低到无抑制剂时的1/4，轻度耐药株(Shhs18)MIC值降低到1/8。KU-60019终浓度为50mM时，高度耐药株(Shjt42b)降低为原1/2。该结果说明Tel1/ATM抑制剂可明显增加烟曲霉对伊曲康唑的敏感性，从侧面印证Tel1参与了烟曲霉唑类耐药。
在KU-60019作用下，无论烟曲霉标准株Af293还是耐药株shjt42b，shjt40或者shhs18对做类药物伊曲康唑ITZ都有显著增效作用，即相应MIC下降。对另外两种唑类药物伏立康唑VRC和泊沙康唑POS，以及费唑类药物两性霉素B(AmB)，则增效不明显。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (4)
1.丝氨酸/苏氨酸蛋白激酶Tel1在制备降低烟曲霉唑类耐药性的药物中的应用。
2.如权利要求1所述的应用，其特征在于，所述药物为有效剂量的抑制丝氨酸/苏氨酸蛋白激酶Tel1/ATM的抑制剂。
3.如权利要求2所述的应用，其特征在于，所述药物为Tel1/ATM抑制剂KU-60019。
4.如权利要求1所述的应用，其特征在于，所述药物包括药物学上可以接受的载体；所述载体为稀释剂、缓冲剂、混悬剂、乳剂、颗粒剂、包囊剂、赋形剂、填充剂、粘合剂、喷雾剂、透皮吸收剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、着色剂、矫味剂或吸附载体。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210839637.4A CN115105596B (zh) | 2022-07-18 | 2022-07-18 | Tel1在制备降低烟曲霉唑类耐药性的药物中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210839637.4A CN115105596B (zh) | 2022-07-18 | 2022-07-18 | Tel1在制备降低烟曲霉唑类耐药性的药物中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115105596A true CN115105596A (zh) | 2022-09-27 |
| CN115105596B CN115105596B (zh) | 2024-01-30 |
Family
ID=83331459
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210839637.4A Active CN115105596B (zh) | 2022-07-18 | 2022-07-18 | Tel1在制备降低烟曲霉唑类耐药性的药物中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115105596B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116077499A (zh) * | 2022-11-28 | 2023-05-09 | 徐州医科大学 | 一种抗耐唑型烟曲霉的药物组合物及其应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5739283A (en) * | 1995-06-07 | 1998-04-14 | The Ohio State Research Foundation | Prevention of invasive pulmonary aspergillosis with serine proteinase inhibitors |
| KR20170077848A (ko) * | 2017-05-22 | 2017-07-06 | (주)앰틱스바이오 | 진균 감염 치료 및 예방을 위한 Bud32 단백질 및 이의 용도 |
| US20180256587A1 (en) * | 2015-09-21 | 2018-09-13 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | A Synthetic Lethal Drug Combination for Treating Renal Cell Carcinoma |
-
2022
- 2022-07-18 CN CN202210839637.4A patent/CN115105596B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5739283A (en) * | 1995-06-07 | 1998-04-14 | The Ohio State Research Foundation | Prevention of invasive pulmonary aspergillosis with serine proteinase inhibitors |
| US20180256587A1 (en) * | 2015-09-21 | 2018-09-13 | Commissariat A L'energie Atomique Et Aux Energies Alternatives | A Synthetic Lethal Drug Combination for Treating Renal Cell Carcinoma |
| KR20170077848A (ko) * | 2017-05-22 | 2017-07-06 | (주)앰틱스바이오 | 진균 감염 치료 및 예방을 위한 Bud32 단백질 및 이의 용도 |
Non-Patent Citations (1)
| Title |
|---|
| 王倩等: "真菌的多向耐药性ABC转运蛋白", 遗传, vol. 33, no. 10, pages 1048 - 1056 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116077499A (zh) * | 2022-11-28 | 2023-05-09 | 徐州医科大学 | 一种抗耐唑型烟曲霉的药物组合物及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115105596B (zh) | 2024-01-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lindsay et al. | Analysis of Candida albicans mutants defective in the Cdk8 module of mediator reveal links between metabolism and biofilm formation | |
| Thakre et al. | Limonene inhibits Candida albicans growth by inducing apoptosis | |
| Li et al. | Delicate metabolic control and coordinated stress response critically determine antifungal tolerance of Candida albicans biofilm persisters | |
| Wurtele et al. | Modulation of histone H3 lysine 56 acetylation as an antifungal therapeutic strategy | |
| Reddy et al. | Endothelial cell peroxisome proliferator–activated receptor γ reduces endotoxemic pulmonary inflammation and injury | |
| Li et al. | Mitochondrial dysfunctions trigger the calcium signaling-dependent fungal multidrug resistance | |
| Wang et al. | Post-translational regulation of autophagy is involved in intra-microbiome suppression of fungal pathogens | |
| Kan et al. | Newcastle-disease-virus-induced ferroptosis through nutrient deprivation and ferritinophagy in tumor cells | |
| Zheng et al. | Inhibition of Cathepsin D (CTSD) enhances radiosensitivity of glioblastoma cells by attenuating autophagy | |
| Ellis et al. | Occidiofungin's chemical stability and in vitro potency against Candida species | |
| Brahimi-Horn et al. | Local mitochondrial-endolysosomal microfusion cleaves voltage-dependent anion channel 1 to promote survival in hypoxia | |
| Du et al. | N-acetylglucosamine-induced cell death in Candida albicans and its implications for adaptive mechanisms of nutrient sensing in yeasts | |
| Dhamgaye et al. | In vitro effect of malachite green on Candida albicans involves multiple pathways and transcriptional regulators UPC2 and STP2 | |
| De Cremer et al. | Stimulation of superoxide production increases fungicidal action of miconazole against Candida albicans biofilms | |
| Armenta et al. | Ferroptosis inhibition by lysosome-dependent catabolism of extracellular protein | |
| Tong et al. | Berberine reverses multidrug resistance in Candida albicans by hijacking the drug efflux pump Mdr1p | |
| Guo et al. | Plagiochin E, a botanic‐derived phenolic compound, reverses fungal resistance to fluconazole relating to the efflux pump | |
| Ben Yaakov et al. | Identification and characterization of haemofungin, a novel antifungal compound that inhibits the final step of haem biosynthesis | |
| Moloney et al. | The iron-responsive microsomal proteome of Aspergillus fumigatus | |
| Xue et al. | Novel mitochondrial complex I-inhibiting peptides restrain NADH dehydrogenase activity | |
| Polonelli et al. | A naturally occurring antibody fragment neutralizes infectivity of diverse infectious agents | |
| Aguiar et al. | Uptake of the siderophore triacetylfusarinine C, but not fusarinine C, is crucial for virulence of Aspergillus fumigatus | |
| Fu et al. | Genetic analysis of Hsp90 function in Cryptococcus neoformans highlights key roles in stress tolerance and virulence | |
| Huang et al. | Antifungal activity and potential mechanism of berberine hydrochloride against fluconazole-resistant Candida albicans | |
| Cai et al. | Deubiquitinase Ubp3 regulates ribophagy and deubiquitinates Smo1 for appressorium‐mediated infection by Magnaporthe oryzae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |