WO1997046250A1 - Methods of modifying feeding behavior, compounds useful in such methods, and dna encoding a hypothalamic atypical neuropeptide y/peptide yy receptor (y5) - Google Patents
Methods of modifying feeding behavior, compounds useful in such methods, and dna encoding a hypothalamic atypical neuropeptide y/peptide yy receptor (y5) Download PDFInfo
- Publication number
- WO1997046250A1 WO1997046250A1 PCT/US1997/009504 US9709504W WO9746250A1 WO 1997046250 A1 WO1997046250 A1 WO 1997046250A1 US 9709504 W US9709504 W US 9709504W WO 9746250 A1 WO9746250 A1 WO 9746250A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- receptor
- human
- chemical compound
- compound
- cell
- Prior art date
Links
- 150000001875 compounds Chemical class 0.000 title claims abstract description 476
- 238000000034 method Methods 0.000 title claims abstract description 285
- 230000004634 feeding behavior Effects 0.000 title claims abstract description 33
- 102400000064 Neuropeptide Y Human genes 0.000 title description 65
- URPYMXQQVHTUDU-OFGSCBOVSA-N nucleopeptide y Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 URPYMXQQVHTUDU-OFGSCBOVSA-N 0.000 title description 55
- 230000002267 hypothalamic effect Effects 0.000 title description 19
- 108010070727 peptide YY receptor Proteins 0.000 title description 3
- 108050002826 Neuropeptide Y Receptor Proteins 0.000 title description 2
- 101710198055 Neuropeptide Y receptor type 5 Proteins 0.000 claims abstract description 561
- 102100029549 Neuropeptide Y receptor type 5 Human genes 0.000 claims abstract description 557
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 92
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 90
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 90
- 239000013598 vector Substances 0.000 claims abstract description 43
- 230000009261 transgenic effect Effects 0.000 claims abstract description 31
- 241001465754 Metazoa Species 0.000 claims abstract description 30
- 239000005557 antagonist Substances 0.000 claims abstract description 22
- 230000001965 increasing effect Effects 0.000 claims abstract description 18
- 108020004711 Nucleic Acid Probes Proteins 0.000 claims abstract description 16
- 239000002853 nucleic acid probe Substances 0.000 claims abstract description 16
- 208000008589 Obesity Diseases 0.000 claims abstract description 15
- 235000020824 obesity Nutrition 0.000 claims abstract description 15
- 239000000556 agonist Substances 0.000 claims abstract description 14
- 206010006550 Bulimia nervosa Diseases 0.000 claims abstract description 11
- 230000000295 complement effect Effects 0.000 claims abstract description 11
- 230000003247 decreasing effect Effects 0.000 claims abstract description 10
- 208000032841 Bulimia Diseases 0.000 claims abstract description 9
- 208000022531 anorexia Diseases 0.000 claims abstract description 8
- 206010061428 decreased appetite Diseases 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 386
- 102000005962 receptors Human genes 0.000 claims description 211
- 108020003175 receptors Proteins 0.000 claims description 211
- 230000027455 binding Effects 0.000 claims description 189
- 241000282414 Homo sapiens Species 0.000 claims description 178
- 101000633401 Homo sapiens Neuropeptide Y receptor type 5 Proteins 0.000 claims description 120
- 241000282465 Canis Species 0.000 claims description 103
- 108020004414 DNA Proteins 0.000 claims description 101
- 239000012528 membrane Substances 0.000 claims description 96
- 230000014509 gene expression Effects 0.000 claims description 86
- 230000000694 effects Effects 0.000 claims description 85
- 230000004913 activation Effects 0.000 claims description 78
- 229940044551 receptor antagonist Drugs 0.000 claims description 58
- 239000002464 receptor antagonist Substances 0.000 claims description 58
- 101000633398 Rattus norvegicus Neuropeptide Y receptor type 5 Proteins 0.000 claims description 55
- 239000003446 ligand Substances 0.000 claims description 55
- 239000002299 complementary DNA Substances 0.000 claims description 53
- 230000004044 response Effects 0.000 claims description 53
- 230000005856 abnormality Effects 0.000 claims description 48
- 108010002245 Neuropeptide Y4 receptor Proteins 0.000 claims description 44
- 102000028435 Neuropeptide Y4 receptor Human genes 0.000 claims description 44
- 150000001413 amino acids Chemical group 0.000 claims description 43
- -1 peptidyl compound Chemical class 0.000 claims description 41
- 108020004999 messenger RNA Proteins 0.000 claims description 40
- 239000008194 pharmaceutical composition Substances 0.000 claims description 39
- 210000004962 mammalian cell Anatomy 0.000 claims description 38
- 239000000523 sample Substances 0.000 claims description 37
- 241000124008 Mammalia Species 0.000 claims description 35
- 230000008569 process Effects 0.000 claims description 35
- 101000652582 Homo sapiens Antigen peptide transporter 2 Proteins 0.000 claims description 34
- 102000054264 human TAP2 Human genes 0.000 claims description 34
- 239000000284 extract Substances 0.000 claims description 32
- 239000013612 plasmid Substances 0.000 claims description 32
- 230000008859 change Effects 0.000 claims description 31
- 125000003729 nucleotide group Chemical group 0.000 claims description 31
- 208000019454 Feeding and Eating disease Diseases 0.000 claims description 30
- 239000012634 fragment Substances 0.000 claims description 30
- 208000018460 Feeding disease Diseases 0.000 claims description 28
- 239000002773 nucleotide Substances 0.000 claims description 28
- 230000009870 specific binding Effects 0.000 claims description 26
- 229940122770 Neurotransmitter uptake inhibitor Drugs 0.000 claims description 25
- 239000003901 neurotransmitter uptake inhibitor Substances 0.000 claims description 25
- 230000001105 regulatory effect Effects 0.000 claims description 25
- 230000007423 decrease Effects 0.000 claims description 23
- XOFLBQFBSOEHOG-UUOKFMHZSA-N γS-GTP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=S)[C@@H](O)[C@H]1O XOFLBQFBSOEHOG-UUOKFMHZSA-N 0.000 claims description 23
- 239000003937 drug carrier Substances 0.000 claims description 22
- 230000002159 abnormal effect Effects 0.000 claims description 20
- 229940121970 Galanin receptor antagonist Drugs 0.000 claims description 17
- 241000238631 Hexapoda Species 0.000 claims description 17
- 239000000126 substance Substances 0.000 claims description 16
- 108091034117 Oligonucleotide Proteins 0.000 claims description 14
- 230000003834 intracellular effect Effects 0.000 claims description 14
- 241000701447 unidentified baculovirus Species 0.000 claims description 14
- 101000603245 Homo sapiens Neuropeptide Y receptor type 2 Proteins 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 12
- 239000011575 calcium Substances 0.000 claims description 12
- 229910052791 calcium Inorganic materials 0.000 claims description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 12
- 108091032973 (ribonucleotides)n+m Proteins 0.000 claims description 11
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 108060000200 adenylate cyclase Proteins 0.000 claims description 9
- 102000030621 adenylate cyclase Human genes 0.000 claims description 9
- 210000000170 cell membrane Anatomy 0.000 claims description 9
- 230000005764 inhibitory process Effects 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 230000001939 inductive effect Effects 0.000 claims description 8
- 108091008146 restriction endonucleases Proteins 0.000 claims description 8
- 238000013519 translation Methods 0.000 claims description 8
- 239000000074 antisense oligonucleotide Substances 0.000 claims description 7
- 238000012230 antisense oligonucleotides Methods 0.000 claims description 7
- 206010037211 Psychomotor hyperactivity Diseases 0.000 claims description 6
- 210000003292 kidney cell Anatomy 0.000 claims description 6
- 230000003542 behavioural effect Effects 0.000 claims description 5
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 5
- 230000004048 modification Effects 0.000 claims description 5
- 238000012986 modification Methods 0.000 claims description 5
- 230000001766 physiological effect Effects 0.000 claims description 5
- DBGIVFWFUFKIQN-VIFPVBQESA-N (+)-Fenfluramine Chemical group CCN[C@@H](C)CC1=CC=CC(C(F)(F)F)=C1 DBGIVFWFUFKIQN-VIFPVBQESA-N 0.000 claims description 4
- 230000009137 competitive binding Effects 0.000 claims description 4
- 229960004597 dexfenfluramine Drugs 0.000 claims description 4
- 230000006801 homologous recombination Effects 0.000 claims description 4
- 238000002744 homologous recombination Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 210000004498 neuroglial cell Anatomy 0.000 claims description 4
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 claims description 4
- UNAANXDKBXWMLN-UHFFFAOYSA-N sibutramine Chemical compound C=1C=C(Cl)C=CC=1C1(C(N(C)C)CC(C)C)CCC1 UNAANXDKBXWMLN-UHFFFAOYSA-N 0.000 claims description 4
- 229960004425 sibutramine Drugs 0.000 claims description 4
- 238000013268 sustained release Methods 0.000 claims description 4
- 239000012730 sustained-release form Substances 0.000 claims description 4
- 210000005253 yeast cell Anatomy 0.000 claims description 4
- DBGIVFWFUFKIQN-UHFFFAOYSA-N (+-)-Fenfluramine Chemical group CCNC(C)CC1=CC=CC(C(F)(F)F)=C1 DBGIVFWFUFKIQN-UHFFFAOYSA-N 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 3
- 102000011392 Galanin receptor Human genes 0.000 claims description 3
- 108050001605 Galanin receptor Proteins 0.000 claims description 3
- 229960001582 fenfluramine Drugs 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 230000001537 neural effect Effects 0.000 claims description 3
- 108020004491 Antisense DNA Proteins 0.000 claims description 2
- 108020005544 Antisense RNA Proteins 0.000 claims description 2
- 241000283073 Equus caballus Species 0.000 claims description 2
- 241000282324 Felis Species 0.000 claims description 2
- 102100036584 Galanin receptor type 2 Human genes 0.000 claims description 2
- 102100036588 Galanin receptor type 3 Human genes 0.000 claims description 2
- 101001072780 Homo sapiens Galanin receptor type 2 Proteins 0.000 claims description 2
- 101001072777 Homo sapiens Galanin receptor type 3 Proteins 0.000 claims description 2
- 239000003816 antisense DNA Substances 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims 1
- 230000002401 inhibitory effect Effects 0.000 claims 1
- 235000012631 food intake Nutrition 0.000 abstract description 20
- 230000000692 anti-sense effect Effects 0.000 abstract description 5
- 241000700159 Rattus Species 0.000 description 111
- 210000004379 membrane Anatomy 0.000 description 86
- 108090000765 processed proteins & peptides Proteins 0.000 description 63
- 101710151321 Melanostatin Proteins 0.000 description 62
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 54
- 102000004196 processed proteins & peptides Human genes 0.000 description 42
- 239000000243 solution Substances 0.000 description 39
- 102100029909 Peptide YY Human genes 0.000 description 37
- 108010088847 Peptide YY Proteins 0.000 description 37
- YNXLOPYTAAFMTN-SBUIBGKBSA-N C([C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)C1=CC=C(O)C=C1 Chemical compound C([C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)C1=CC=C(O)C=C1 YNXLOPYTAAFMTN-SBUIBGKBSA-N 0.000 description 33
- 239000000018 receptor agonist Substances 0.000 description 33
- 229940044601 receptor agonist Drugs 0.000 description 33
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 31
- 108090000623 proteins and genes Proteins 0.000 description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 30
- 239000002287 radioligand Substances 0.000 description 28
- 229910001868 water Inorganic materials 0.000 description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 22
- 229920001184 polypeptide Polymers 0.000 description 22
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 21
- 239000000047 product Substances 0.000 description 21
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 20
- 108010052285 Membrane Proteins Proteins 0.000 description 20
- 230000000875 corresponding effect Effects 0.000 description 20
- 238000006073 displacement reaction Methods 0.000 description 20
- 229940079593 drug Drugs 0.000 description 20
- 239000003814 drug Substances 0.000 description 20
- 102000018697 Membrane Proteins Human genes 0.000 description 19
- 238000002474 experimental method Methods 0.000 description 19
- 230000037406 food intake Effects 0.000 description 19
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 19
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 19
- 210000004556 brain Anatomy 0.000 description 18
- 239000013615 primer Substances 0.000 description 18
- 239000011541 reaction mixture Substances 0.000 description 18
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 16
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 16
- 238000009396 hybridization Methods 0.000 description 16
- 210000004940 nucleus Anatomy 0.000 description 16
- 238000012360 testing method Methods 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 16
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 15
- 230000000144 pharmacologic effect Effects 0.000 description 15
- 238000000159 protein binding assay Methods 0.000 description 15
- 108091026890 Coding region Proteins 0.000 description 14
- 102100038991 Neuropeptide Y receptor type 2 Human genes 0.000 description 14
- 210000003016 hypothalamus Anatomy 0.000 description 14
- 101500025005 Homo sapiens Neuropeptide Y Proteins 0.000 description 13
- 235000013305 food Nutrition 0.000 description 13
- 102000004169 proteins and genes Human genes 0.000 description 13
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 12
- 101710197945 Neuropeptide Y receptor type 2 Proteins 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 238000012216 screening Methods 0.000 description 12
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 11
- 102000018886 Pancreatic Polypeptide Human genes 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 11
- 235000001014 amino acid Nutrition 0.000 description 11
- 230000002860 competitive effect Effects 0.000 description 11
- 235000019439 ethyl acetate Nutrition 0.000 description 11
- 229910052757 nitrogen Inorganic materials 0.000 description 11
- 239000008188 pellet Substances 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 11
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Inorganic materials [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 11
- 239000007787 solid Substances 0.000 description 11
- HFDKKNHCYWNNNQ-YOGANYHLSA-N 75976-10-2 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)N)C(C)C)[C@@H](C)O)C1=CC=C(O)C=C1 HFDKKNHCYWNNNQ-YOGANYHLSA-N 0.000 description 10
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 10
- 101000983124 Sus scrofa Pancreatic prohormone precursor Proteins 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 239000012148 binding buffer Substances 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 238000002825 functional assay Methods 0.000 description 10
- 239000007924 injection Substances 0.000 description 10
- 238000002347 injection Methods 0.000 description 10
- 238000000611 regression analysis Methods 0.000 description 10
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 9
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 9
- 229940093499 ethyl acetate Drugs 0.000 description 9
- 238000004992 fast atom bombardment mass spectroscopy Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 238000002844 melting Methods 0.000 description 9
- 230000008018 melting Effects 0.000 description 9
- 230000009871 nonspecific binding Effects 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 239000003981 vehicle Substances 0.000 description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 8
- PAYRUJLWNCNPSJ-UHFFFAOYSA-N Aniline Chemical compound NC1=CC=CC=C1 PAYRUJLWNCNPSJ-UHFFFAOYSA-N 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 8
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 8
- 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 8
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 8
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 8
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 8
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 8
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 8
- 238000012217 deletion Methods 0.000 description 8
- 230000037430 deletion Effects 0.000 description 8
- 238000001914 filtration Methods 0.000 description 8
- 230000000971 hippocampal effect Effects 0.000 description 8
- 210000001320 hippocampus Anatomy 0.000 description 8
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 230000002829 reductive effect Effects 0.000 description 8
- 238000000527 sonication Methods 0.000 description 8
- 239000007858 starting material Substances 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 7
- 238000000636 Northern blotting Methods 0.000 description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- 241000269370 Xenopus <genus> Species 0.000 description 7
- 150000001412 amines Chemical class 0.000 description 7
- 229910052799 carbon Inorganic materials 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- 208000035475 disorder Diseases 0.000 description 7
- 238000002955 isolation Methods 0.000 description 7
- 244000005700 microbiome Species 0.000 description 7
- DWZUVGJMKKOCKN-XLUSCKSJSA-N npy2-36, porcine Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H]1NCCC1)C1=CC=C(O)C=C1 DWZUVGJMKKOCKN-XLUSCKSJSA-N 0.000 description 7
- 230000036961 partial effect Effects 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 239000000741 silica gel Substances 0.000 description 7
- 229910002027 silica gel Inorganic materials 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 6
- 108020004635 Complementary DNA Proteins 0.000 description 6
- 229920002307 Dextran Polymers 0.000 description 6
- 101000983116 Homo sapiens Pancreatic prohormone Proteins 0.000 description 6
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 6
- 229930182555 Penicillin Natural products 0.000 description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- 241000700605 Viruses Species 0.000 description 6
- 239000011543 agarose gel Substances 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 6
- 239000001110 calcium chloride Substances 0.000 description 6
- 229910001628 calcium chloride Inorganic materials 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 239000013592 cell lysate Substances 0.000 description 6
- 238000010367 cloning Methods 0.000 description 6
- 229960002086 dextran Drugs 0.000 description 6
- 229960004132 diethyl ether Drugs 0.000 description 6
- 235000013601 eggs Nutrition 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 210000002569 neuron Anatomy 0.000 description 6
- HEALDAASUSTTPJ-QGTLAHJGSA-N npy13-36, porcine Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H]1NCCC1)C1=CC=C(O)C=C1 HEALDAASUSTTPJ-QGTLAHJGSA-N 0.000 description 6
- 239000003921 oil Substances 0.000 description 6
- 210000000287 oocyte Anatomy 0.000 description 6
- 229940049954 penicillin Drugs 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 description 6
- 235000011152 sodium sulphate Nutrition 0.000 description 6
- 229960005322 streptomycin Drugs 0.000 description 6
- AKKYWYCANLVLAS-UHFFFAOYSA-N 2-chloro-n-phenylquinazolin-4-amine Chemical compound C=12C=CC=CC2=NC(Cl)=NC=1NC1=CC=CC=C1 AKKYWYCANLVLAS-UHFFFAOYSA-N 0.000 description 5
- QBWKPGNFQQJGFY-QLFBSQMISA-N 3-[(1r)-1-[(2r,6s)-2,6-dimethylmorpholin-4-yl]ethyl]-n-[6-methyl-3-(1h-pyrazol-4-yl)imidazo[1,2-a]pyrazin-8-yl]-1,2-thiazol-5-amine Chemical compound N1([C@H](C)C2=NSC(NC=3C4=NC=C(N4C=C(C)N=3)C3=CNN=C3)=C2)C[C@H](C)O[C@H](C)C1 QBWKPGNFQQJGFY-QLFBSQMISA-N 0.000 description 5
- 108020004705 Codon Proteins 0.000 description 5
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 5
- 108091006027 G proteins Proteins 0.000 description 5
- 102000030782 GTP binding Human genes 0.000 description 5
- 108091000058 GTP-Binding Proteins 0.000 description 5
- 239000007836 KH2PO4 Substances 0.000 description 5
- 101500025021 Rattus norvegicus Neuropeptide Y Proteins 0.000 description 5
- 238000002105 Southern blotting Methods 0.000 description 5
- 229940098773 bovine serum albumin Drugs 0.000 description 5
- 244000309466 calf Species 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 5
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 5
- 229940125846 compound 25 Drugs 0.000 description 5
- 238000002425 crystallisation Methods 0.000 description 5
- 230000008025 crystallization Effects 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 235000019441 ethanol Nutrition 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 5
- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 5
- 230000027425 release of sequestered calcium ion into cytosol Effects 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 239000013589 supplement Substances 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 229940086542 triethylamine Drugs 0.000 description 5
- QCQCHGYLTSGIGX-GHXANHINSA-N 4-[[(3ar,5ar,5br,7ar,9s,11ar,11br,13as)-5a,5b,8,8,11a-pentamethyl-3a-[(5-methylpyridine-3-carbonyl)amino]-2-oxo-1-propan-2-yl-4,5,6,7,7a,9,10,11,11b,12,13,13a-dodecahydro-3h-cyclopenta[a]chrysen-9-yl]oxy]-2,2-dimethyl-4-oxobutanoic acid Chemical compound N([C@@]12CC[C@@]3(C)[C@]4(C)CC[C@H]5C(C)(C)[C@@H](OC(=O)CC(C)(C)C(O)=O)CC[C@]5(C)[C@H]4CC[C@@H]3C1=C(C(C2)=O)C(C)C)C(=O)C1=CN=CC(C)=C1 QCQCHGYLTSGIGX-GHXANHINSA-N 0.000 description 4
- QIVBCDIJIAJPQS-SECBINFHSA-N D-tryptophane Chemical compound C1=CC=C2C(C[C@@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-SECBINFHSA-N 0.000 description 4
- 206010011953 Decreased activity Diseases 0.000 description 4
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 4
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 4
- 239000004743 Polypropylene Substances 0.000 description 4
- 101000983122 Rattus norvegicus Pancreatic prohormone Proteins 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 150000001408 amides Chemical class 0.000 description 4
- 238000013459 approach Methods 0.000 description 4
- KUWBXRGRMQZCSS-HSZRJFAPSA-N bibp-3226 Chemical compound N([C@H](CCCN=C(N)N)C(=O)NCC=1C=CC(O)=CC=1)C(=O)C(C=1C=CC=CC=1)C1=CC=CC=C1 KUWBXRGRMQZCSS-HSZRJFAPSA-N 0.000 description 4
- 239000012267 brine Substances 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 238000012512 characterization method Methods 0.000 description 4
- 238000004440 column chromatography Methods 0.000 description 4
- 239000013078 crystal Substances 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 239000000839 emulsion Substances 0.000 description 4
- 238000011067 equilibration Methods 0.000 description 4
- 238000001400 expression cloning Methods 0.000 description 4
- 210000002950 fibroblast Anatomy 0.000 description 4
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 4
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 4
- 229960001340 histamine Drugs 0.000 description 4
- 230000004807 localization Effects 0.000 description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 description 4
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 4
- 210000003574 melanophore Anatomy 0.000 description 4
- 239000000155 melt Substances 0.000 description 4
- 239000002751 oligonucleotide probe Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000026731 phosphorylation Effects 0.000 description 4
- 238000006366 phosphorylation reaction Methods 0.000 description 4
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000003127 radioimmunoassay Methods 0.000 description 4
- 229910000029 sodium carbonate Inorganic materials 0.000 description 4
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 4
- DYHSDKLCOJIUFX-UHFFFAOYSA-N tert-butoxycarbonyl anhydride Chemical compound CC(C)(C)OC(=O)OC(=O)OC(C)(C)C DYHSDKLCOJIUFX-UHFFFAOYSA-N 0.000 description 4
- NYXOBVVHJZENCO-UHFFFAOYSA-N tert-butyl n-[[4-(aminomethyl)cyclohexyl]methyl]carbamate Chemical compound CC(C)(C)OC(=O)NCC1CCC(CN)CC1 NYXOBVVHJZENCO-UHFFFAOYSA-N 0.000 description 4
- 230000000542 thalamic effect Effects 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- AOSZTAHDEDLTLQ-AZKQZHLXSA-N (1S,2S,4R,8S,9S,11S,12R,13S,19S)-6-[(3-chlorophenyl)methyl]-12,19-difluoro-11-hydroxy-8-(2-hydroxyacetyl)-9,13-dimethyl-6-azapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17-dien-16-one Chemical compound C([C@@H]1C[C@H]2[C@H]3[C@]([C@]4(C=CC(=O)C=C4[C@@H](F)C3)C)(F)[C@@H](O)C[C@@]2([C@@]1(C1)C(=O)CO)C)N1CC1=CC=CC(Cl)=C1 AOSZTAHDEDLTLQ-AZKQZHLXSA-N 0.000 description 3
- NTAGXJQHJQUOOA-UHFFFAOYSA-N 1,2,3,4-tetrahydronaphthalene-2-carboxylic acid Chemical compound C1=CC=C2CC(C(=O)O)CCC2=C1 NTAGXJQHJQUOOA-UHFFFAOYSA-N 0.000 description 3
- TUQSVSYUEBNNKQ-UHFFFAOYSA-N 2,4-dichloroquinazoline Chemical compound C1=CC=CC2=NC(Cl)=NC(Cl)=C21 TUQSVSYUEBNNKQ-UHFFFAOYSA-N 0.000 description 3
- QVRRAZDRHLCWTR-UHFFFAOYSA-N 2-chloro-6-methoxy-n-phenylquinazolin-4-amine Chemical compound C12=CC(OC)=CC=C2N=C(Cl)N=C1NC1=CC=CC=C1 QVRRAZDRHLCWTR-UHFFFAOYSA-N 0.000 description 3
- RJWNGTJWTULKJW-UHFFFAOYSA-N 2-chloro-8-methoxy-n-phenylquinazolin-4-amine Chemical compound N1=C(Cl)N=C2C(OC)=CC=CC2=C1NC1=CC=CC=C1 RJWNGTJWTULKJW-UHFFFAOYSA-N 0.000 description 3
- AZEKNJGFCSHZID-UHFFFAOYSA-N 4-[[(2-methylpropan-2-yl)oxycarbonylamino]methyl]cyclohexane-1-carboxylic acid Chemical compound CC(C)(C)OC(=O)NCC1CCC(C(O)=O)CC1 AZEKNJGFCSHZID-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- ZYZHIGVFJMKISJ-XYPYZODXSA-N CC(C)(C)OC(=O)NC[C@H]1CC[C@H](CN=[N+]=[N-])CC1 Chemical compound CC(C)(C)OC(=O)NC[C@H]1CC[C@H](CN=[N+]=[N-])CC1 ZYZHIGVFJMKISJ-XYPYZODXSA-N 0.000 description 3
- 229940126657 Compound 17 Drugs 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- UQABYHGXWYXDTK-UUOKFMHZSA-N GppNP Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)NP(O)(O)=O)[C@@H](O)[C@H]1O UQABYHGXWYXDTK-UUOKFMHZSA-N 0.000 description 3
- 229930182816 L-glutamine Natural products 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000001464 adherent effect Effects 0.000 description 3
- 229910052786 argon Inorganic materials 0.000 description 3
- 235000010323 ascorbic acid Nutrition 0.000 description 3
- 239000011668 ascorbic acid Substances 0.000 description 3
- 239000012298 atmosphere Substances 0.000 description 3
- 230000001593 cAMP accumulation Effects 0.000 description 3
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012230 colorless oil Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 235000005686 eating Nutrition 0.000 description 3
- 239000012894 fetal calf serum Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000003306 harvesting Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 238000000185 intracerebroventricular administration Methods 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 239000007758 minimum essential medium Substances 0.000 description 3
- XKWLBZBHXDNBTH-UHFFFAOYSA-N n-[[4-(aminomethyl)cyclohexyl]methyl]naphthalene-1-sulfonamide Chemical compound C1CC(CN)CCC1CNS(=O)(=O)C1=CC=CC2=CC=CC=C12 XKWLBZBHXDNBTH-UHFFFAOYSA-N 0.000 description 3
- 210000002963 paraventricular hypothalamic nucleus Anatomy 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 3
- 238000001953 recrystallisation Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000010992 reflux Methods 0.000 description 3
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 210000003523 substantia nigra Anatomy 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 239000012134 supernatant fraction Substances 0.000 description 3
- KSXPGASLEFGROT-UHFFFAOYSA-N tert-butyl n-[[4-(hydroxymethyl)cyclohexyl]methyl]carbamate Chemical compound CC(C)(C)OC(=O)NCC1CCC(CO)CC1 KSXPGASLEFGROT-UHFFFAOYSA-N 0.000 description 3
- XCPXHKPEXDRGKP-UHFFFAOYSA-N tert-butyl n-[[4-[(naphthalen-1-ylsulfonylamino)methyl]cyclohexyl]methyl]carbamate Chemical compound C1CC(CNC(=O)OC(C)(C)C)CCC1CNS(=O)(=O)C1=CC=CC2=CC=CC=C12 XCPXHKPEXDRGKP-UHFFFAOYSA-N 0.000 description 3
- 150000003512 tertiary amines Chemical class 0.000 description 3
- 238000004809 thin layer chromatography Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- CUKWUWBLQQDQAC-VEQWQPCFSA-N (3s)-3-amino-4-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(2s)-1-[(2s)-2-[[(1s)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-ox Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 CUKWUWBLQQDQAC-VEQWQPCFSA-N 0.000 description 2
- CIWNHTXCBHTWRV-UHFFFAOYSA-N 1-(4-aminophenyl)-n-methylmethanesulfonamide Chemical compound CNS(=O)(=O)CC1=CC=C(N)C=C1 CIWNHTXCBHTWRV-UHFFFAOYSA-N 0.000 description 2
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical compound CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- YPNGRZOYTLZRBD-UHFFFAOYSA-N 2-chloroquinazolin-4-amine Chemical compound C1=CC=C2C(N)=NC(Cl)=NC2=C1 YPNGRZOYTLZRBD-UHFFFAOYSA-N 0.000 description 2
- CZMRCDWAGMRECN-UHFFFAOYSA-N 2-{[3,4-dihydroxy-2,5-bis(hydroxymethyl)oxolan-2-yl]oxy}-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound OCC1OC(CO)(OC2OC(CO)C(O)C(O)C2O)C(O)C1O CZMRCDWAGMRECN-UHFFFAOYSA-N 0.000 description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 2
- ISQQHOXHAUDCAK-UHFFFAOYSA-N 4-n-phenyl-2-n-(4-piperidin-1-ylphenyl)quinazoline-2,4-diamine;dihydrochloride Chemical compound Cl.Cl.C1CCCCN1C(C=C1)=CC=C1NC1=NC(NC=2C=CC=CC=2)=C(C=CC=C2)C2=N1 ISQQHOXHAUDCAK-UHFFFAOYSA-N 0.000 description 2
- 102000040125 5-hydroxytryptamine receptor family Human genes 0.000 description 2
- 108091032151 5-hydroxytryptamine receptor family Proteins 0.000 description 2
- 108060003345 Adrenergic Receptor Proteins 0.000 description 2
- 102000017910 Adrenergic receptor Human genes 0.000 description 2
- 208000000044 Amnesia Diseases 0.000 description 2
- 102400000345 Angiotensin-2 Human genes 0.000 description 2
- 101800000733 Angiotensin-2 Proteins 0.000 description 2
- 208000019901 Anxiety disease Diseases 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- ZLJYERMFRNCARL-SHTZXODSSA-N C1C[C@@H](CN)CC[C@@H]1CNS(=O)(=O)C1=CC=C(C=CC=C2)C2=C1 Chemical compound C1C[C@@H](CN)CC[C@@H]1CNS(=O)(=O)C1=CC=C(C=CC=C2)C2=C1 ZLJYERMFRNCARL-SHTZXODSSA-N 0.000 description 2
- 101000633380 Canis lupus familiaris Neuropeptide Y receptor type 5 Proteins 0.000 description 2
- 241001631457 Cannula Species 0.000 description 2
- XDBHXXIAZRUHRJ-UHFFFAOYSA-N Cl.COC(=O)CCC1=CC=C(Cl)C=C1 Chemical compound Cl.COC(=O)CCC1=CC=C(Cl)C=C1 XDBHXXIAZRUHRJ-UHFFFAOYSA-N 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- 206010010904 Convulsion Diseases 0.000 description 2
- 241001559589 Cullen Species 0.000 description 2
- 239000003155 DNA primer Substances 0.000 description 2
- 239000003298 DNA probe Substances 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 101000887490 Homo sapiens Guanine nucleotide-binding protein G(z) subunit alpha Proteins 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-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
- 208000026139 Memory disease Diseases 0.000 description 2
- 208000019695 Migraine disease Diseases 0.000 description 2
- JLTDJTHDQAWBAV-UHFFFAOYSA-N N,N-dimethylaniline Chemical compound CN(C)C1=CC=CC=C1 JLTDJTHDQAWBAV-UHFFFAOYSA-N 0.000 description 2
- 102000028582 Neuropeptide Y5 receptor Human genes 0.000 description 2
- 108010046593 Neuropeptide Y5 receptor Proteins 0.000 description 2
- UGMDUPQJIMCKQL-JHNFVTJISA-N O=C([C@@H]1CC[C@@H](CNC(=O)OC(C)(C)C)CC1)NC(C(=O)OC)CC1=CC=C(Cl)C=C1 Chemical compound O=C([C@@H]1CC[C@@H](CNC(=O)OC(C)(C)C)CC1)NC(C(=O)OC)CC1=CC=C(Cl)C=C1 UGMDUPQJIMCKQL-JHNFVTJISA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 108020004518 RNA Probes Proteins 0.000 description 2
- 239000003391 RNA probe Substances 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 210000004727 amygdala Anatomy 0.000 description 2
- 229950006323 angiotensin ii Drugs 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000036506 anxiety Effects 0.000 description 2
- 229940072107 ascorbate Drugs 0.000 description 2
- 238000000376 autoradiography Methods 0.000 description 2
- HUMNYLRZRPPJDN-UHFFFAOYSA-N benzaldehyde Chemical compound O=CC1=CC=CC=C1 HUMNYLRZRPPJDN-UHFFFAOYSA-N 0.000 description 2
- UORVGPXVDQYIDP-UHFFFAOYSA-N borane Chemical compound B UORVGPXVDQYIDP-UHFFFAOYSA-N 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 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
- 210000001159 caudate nucleus Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 210000001638 cerebellum Anatomy 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 2
- 238000012761 co-transfection Methods 0.000 description 2
- 229940125904 compound 1 Drugs 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 210000000877 corpus callosum Anatomy 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 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 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 description 2
- 238000002651 drug therapy Methods 0.000 description 2
- 210000003890 endocrine cell Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 210000001652 frontal lobe Anatomy 0.000 description 2
- 238000000265 homogenisation Methods 0.000 description 2
- 102000052301 human GNAZ Human genes 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- 230000005291 magnetic effect Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000006984 memory degeneration Effects 0.000 description 2
- 208000023060 memory loss Diseases 0.000 description 2
- YECBIJXISLIIDS-UHFFFAOYSA-N mepyramine Chemical compound C1=CC(OC)=CC=C1CN(CCN(C)C)C1=CC=CC=N1 YECBIJXISLIIDS-UHFFFAOYSA-N 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 2
- 206010027599 migraine Diseases 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical class CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- DASJFYAPNPUBGG-UHFFFAOYSA-N naphthalene-1-sulfonyl chloride Chemical compound C1=CC=C2C(S(=O)(=O)Cl)=CC=CC2=C1 DASJFYAPNPUBGG-UHFFFAOYSA-N 0.000 description 2
- OPECTNGATDYLSS-UHFFFAOYSA-N naphthalene-2-sulfonyl chloride Chemical compound C1=CC=CC2=CC(S(=O)(=O)Cl)=CC=C21 OPECTNGATDYLSS-UHFFFAOYSA-N 0.000 description 2
- 210000000653 nervous system Anatomy 0.000 description 2
- BPGXUIVWLQTVLZ-OFGSCBOVSA-N neuropeptide y(npy) Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 BPGXUIVWLQTVLZ-OFGSCBOVSA-N 0.000 description 2
- 210000000869 occipital lobe Anatomy 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 230000036407 pain Effects 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 125000001151 peptidyl group Chemical group 0.000 description 2
- 210000002509 periaqueductal gray Anatomy 0.000 description 2
- 239000008389 polyethoxylated castor oil Substances 0.000 description 2
- 229920001155 polypropylene Polymers 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 210000002637 putamen Anatomy 0.000 description 2
- 238000003653 radioligand binding assay Methods 0.000 description 2
- 239000000376 reactant Substances 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 108010035889 salmon pancreatic polypeptide Proteins 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 208000019116 sleep disease Diseases 0.000 description 2
- 208000022925 sleep disturbance Diseases 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- 210000000278 spinal cord Anatomy 0.000 description 2
- 238000013222 sprague-dawley male rat Methods 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 210000004281 subthalamic nucleus Anatomy 0.000 description 2
- 210000003863 superior colliculi Anatomy 0.000 description 2
- 238000010189 synthetic method Methods 0.000 description 2
- 210000003478 temporal lobe Anatomy 0.000 description 2
- 210000001103 thalamus Anatomy 0.000 description 2
- ZFXYFBGIUFBOJW-UHFFFAOYSA-N theophylline Chemical compound O=C1N(C)C(=O)N(C)C2=C1NC=N2 ZFXYFBGIUFBOJW-UHFFFAOYSA-N 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical group CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000003146 transient transfection Methods 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 208000016261 weight loss Diseases 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- ZZJYIKPMDIWRSN-TZBSWOFLSA-N (+)-butaclamol Chemical compound C12=CC=CC=C2CCC2=CC=CC3=C2[C@@H]1CN1CC[C@@](C(C)(C)C)(O)C[C@@H]13 ZZJYIKPMDIWRSN-TZBSWOFLSA-N 0.000 description 1
- UAOUIVVJBYDFKD-XKCDOFEDSA-N (1R,9R,10S,11R,12R,15S,18S,21R)-10,11,21-trihydroxy-8,8-dimethyl-14-methylidene-4-(prop-2-enylamino)-20-oxa-5-thia-3-azahexacyclo[9.7.2.112,15.01,9.02,6.012,18]henicosa-2(6),3-dien-13-one Chemical compound C([C@@H]1[C@@H](O)[C@@]23C(C1=C)=O)C[C@H]2[C@]12C(N=C(NCC=C)S4)=C4CC(C)(C)[C@H]1[C@H](O)[C@]3(O)OC2 UAOUIVVJBYDFKD-XKCDOFEDSA-N 0.000 description 1
- QFLWZFQWSBQYPS-AWRAUJHKSA-N (3S)-3-[[(2S)-2-[[(2S)-2-[5-[(3aS,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-3-methylbutanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-[1-bis(4-chlorophenoxy)phosphorylbutylamino]-4-oxobutanoic acid Chemical compound CCCC(NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](Cc1ccc(O)cc1)NC(=O)[C@@H](NC(=O)CCCCC1SC[C@@H]2NC(=O)N[C@H]12)C(C)C)P(=O)(Oc1ccc(Cl)cc1)Oc1ccc(Cl)cc1 QFLWZFQWSBQYPS-AWRAUJHKSA-N 0.000 description 1
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 1
- UAFHRUBCOQPFFM-UHFFFAOYSA-N 1-(aminomethyl)cyclohexane-1-carboxylic acid Chemical compound NCC1(C(O)=O)CCCCC1 UAFHRUBCOQPFFM-UHFFFAOYSA-N 0.000 description 1
- WDYLZDNLDMVHPS-UHFFFAOYSA-N 1-[4-[(4-anilino-6-methoxyquinazolin-2-yl)amino]phenyl]-n-methylmethanesulfonamide Chemical compound C1=CC(CS(=O)(=O)NC)=CC=C1NC1=NC(NC=2C=CC=CC=2)=C(C=C(OC)C=C2)C2=N1 WDYLZDNLDMVHPS-UHFFFAOYSA-N 0.000 description 1
- UBZVTHYHPZOMQT-UHFFFAOYSA-N 1-[4-[(4-anilino-6-methoxyquinazolin-2-yl)amino]phenyl]-n-methylmethanesulfonamide;hydrochloride Chemical compound Cl.C1=CC(CS(=O)(=O)NC)=CC=C1NC1=NC(NC=2C=CC=CC=2)=C(C=C(OC)C=C2)C2=N1 UBZVTHYHPZOMQT-UHFFFAOYSA-N 0.000 description 1
- PTPIKRSQILMMHX-UHFFFAOYSA-N 1-[4-[(4-anilinoquinazolin-2-yl)amino]phenyl]-n-methylmethanesulfonamide;hydrochloride Chemical compound Cl.C1=CC(CS(=O)(=O)NC)=CC=C1NC1=NC(NC=2C=CC=CC=2)=C(C=CC=C2)C2=N1 PTPIKRSQILMMHX-UHFFFAOYSA-N 0.000 description 1
- YDDXVAXDYKBWDX-UHFFFAOYSA-N 1-cyano-3-[2-[[2-(diaminomethylideneamino)-4-thiazolyl]methylthio]ethyl]-2-methylguanidine Chemical compound N#CNC(=NC)NCCSCC1=CSC(N=C(N)N)=N1 YDDXVAXDYKBWDX-UHFFFAOYSA-N 0.000 description 1
- SDQJTWBNWQABLE-UHFFFAOYSA-N 1h-quinazoline-2,4-dione Chemical compound C1=CC=C2C(=O)NC(=O)NC2=C1 SDQJTWBNWQABLE-UHFFFAOYSA-N 0.000 description 1
- WEAMQTSRMCCGSJ-UHFFFAOYSA-N 2,4-dichloro-6-methoxyquinazoline Chemical compound N1=C(Cl)N=C(Cl)C2=CC(OC)=CC=C21 WEAMQTSRMCCGSJ-UHFFFAOYSA-N 0.000 description 1
- DZNPOCHJCGTGCP-UHFFFAOYSA-N 2,4-dichloro-8-methoxyquinazoline Chemical compound N1=C(Cl)N=C2C(OC)=CC=CC2=C1Cl DZNPOCHJCGTGCP-UHFFFAOYSA-N 0.000 description 1
- GRKYINPDAFNVFU-UHFFFAOYSA-N 2-(naphthalen-1-ylamino)-3-phenylpropanenitrile Chemical compound C=1C=CC2=CC=CC=C2C=1NC(C#N)CC1=CC=CC=C1 GRKYINPDAFNVFU-UHFFFAOYSA-N 0.000 description 1
- YSUIQYOGTINQIN-UZFYAQMZSA-N 2-amino-9-[(1S,6R,8R,9S,10R,15R,17R,18R)-8-(6-aminopurin-9-yl)-9,18-difluoro-3,12-dihydroxy-3,12-bis(sulfanylidene)-2,4,7,11,13,16-hexaoxa-3lambda5,12lambda5-diphosphatricyclo[13.2.1.06,10]octadecan-17-yl]-1H-purin-6-one Chemical compound NC1=NC2=C(N=CN2[C@@H]2O[C@@H]3COP(S)(=O)O[C@@H]4[C@@H](COP(S)(=O)O[C@@H]2[C@@H]3F)O[C@H]([C@H]4F)N2C=NC3=C2N=CN=C3N)C(=O)N1 YSUIQYOGTINQIN-UZFYAQMZSA-N 0.000 description 1
- TVTJUIAKQFIXCE-HUKYDQBMSA-N 2-amino-9-[(2R,3S,4S,5R)-4-fluoro-3-hydroxy-5-(hydroxymethyl)oxolan-2-yl]-7-prop-2-ynyl-1H-purine-6,8-dione Chemical compound NC=1NC(C=2N(C(N(C=2N=1)[C@@H]1O[C@@H]([C@H]([C@H]1O)F)CO)=O)CC#C)=O TVTJUIAKQFIXCE-HUKYDQBMSA-N 0.000 description 1
- LNRXUPKGVFICQP-UHFFFAOYSA-N 2-n,4-n-diphenylquinazoline-2,4-diamine;hydrochloride Chemical compound Cl.N=1C(NC=2C=CC=CC=2)=C2C=CC=CC2=NC=1NC1=CC=CC=C1 LNRXUPKGVFICQP-UHFFFAOYSA-N 0.000 description 1
- UWFMZXLJKUNCLI-UHFFFAOYSA-N 2-naphthalen-1-yl-3-phenylpropane-1,2-diamine Chemical compound C=1C=CC2=CC=CC=C2C=1C(N)(CN)CC1=CC=CC=C1 UWFMZXLJKUNCLI-UHFFFAOYSA-N 0.000 description 1
- OFCNTYBPPAQCRE-UHFFFAOYSA-N 3-(2-aminoethyl)-3h-indol-5-ol Chemical compound C1=C(O)C=C2C(CCN)C=NC2=C1 OFCNTYBPPAQCRE-UHFFFAOYSA-N 0.000 description 1
- IMLXLGZJLAOKJN-UHFFFAOYSA-N 4-aminocyclohexan-1-ol Chemical compound NC1CCC(O)CC1 IMLXLGZJLAOKJN-UHFFFAOYSA-N 0.000 description 1
- TVOSOIXYPHKEAR-UHFFFAOYSA-N 4-piperidin-1-ylaniline Chemical compound C1=CC(N)=CC=C1N1CCCCC1 TVOSOIXYPHKEAR-UHFFFAOYSA-N 0.000 description 1
- YYROPELSRYBVMQ-UHFFFAOYSA-N 4-toluenesulfonyl chloride Chemical compound CC1=CC=C(S(Cl)(=O)=O)C=C1 YYROPELSRYBVMQ-UHFFFAOYSA-N 0.000 description 1
- YKKDPYCIXOHMGK-UHFFFAOYSA-N 8-methoxy-2-n-(4-methoxyphenyl)-4-n-phenylquinazoline-2,4-diamine;dihydrochloride Chemical compound Cl.Cl.C1=CC(OC)=CC=C1NC1=NC(NC=2C=CC=CC=2)=C(C=CC=C2OC)C2=N1 YKKDPYCIXOHMGK-UHFFFAOYSA-N 0.000 description 1
- PTWGQXOXPYOLEU-UHFFFAOYSA-N 8-methoxy-2-n-(4-methoxyphenyl)-4-n-phenylquinazoline-2,4-diamine;hydrochloride Chemical compound Cl.C1=CC(OC)=CC=C1NC1=NC(NC=2C=CC=CC=2)=C(C=CC=C2OC)C2=N1 PTWGQXOXPYOLEU-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical class [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000027559 Appetite disease Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108010001478 Bacitracin Proteins 0.000 description 1
- 101000585591 Bos taurus Pancreatic prohormone Proteins 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 101800001415 Bri23 peptide Proteins 0.000 description 1
- 102400000107 C-terminal peptide Human genes 0.000 description 1
- 101800000655 C-terminal peptide Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 241001227713 Chiron Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- UFXOOCKYSBBYFD-YHSIFKNPSA-N Cl.CC(=O)O[C@H]1CC[C@@H](CC1)Nc1nc(Nc2ccccc2)c2ccccc2n1 Chemical compound Cl.CC(=O)O[C@H]1CC[C@@H](CC1)Nc1nc(Nc2ccccc2)c2ccccc2n1 UFXOOCKYSBBYFD-YHSIFKNPSA-N 0.000 description 1
- DIQDKUNCSVFGHH-VPLSKCCHSA-N Cl.Nc1nc(NC[C@H]2CC[C@H](CNS(=O)(=O)c3cccc4ccccc34)CC2)nc2ccccc12 Chemical compound Cl.Nc1nc(NC[C@H]2CC[C@H](CNS(=O)(=O)c3cccc4ccccc34)CC2)nc2ccccc12 DIQDKUNCSVFGHH-VPLSKCCHSA-N 0.000 description 1
- 241001573498 Compacta Species 0.000 description 1
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 1
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 1
- 102000004654 Cyclic GMP-Dependent Protein Kinases Human genes 0.000 description 1
- 108010003591 Cyclic GMP-Dependent Protein Kinases Proteins 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108020003215 DNA Probes Proteins 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 241001269524 Dura Species 0.000 description 1
- 208000030814 Eating disease Diseases 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 229920001917 Ficoll Polymers 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010078321 Guanylate Cyclase Proteins 0.000 description 1
- 102000014469 Guanylate cyclase Human genes 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 101000865224 Homo sapiens D(3) dopamine receptor Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 108700002232 Immediate-Early Genes Proteins 0.000 description 1
- 241000283891 Kobus Species 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
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 229910010084 LiAlH4 Inorganic materials 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- UEQUQVLFIPOEMF-UHFFFAOYSA-N Mianserin Chemical compound C1C2=CC=CC=C2N2CCN(C)CC2C2=CC=CC=C21 UEQUQVLFIPOEMF-UHFFFAOYSA-N 0.000 description 1
- 208000019022 Mood disease Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- FVJRBJIENDRNBE-UHFFFAOYSA-N N-(6-aminohexyl)-1-naphthalenesulfonamide Chemical compound C1=CC=C2C(S(=O)(=O)NCCCCCCN)=CC=CC2=C1 FVJRBJIENDRNBE-UHFFFAOYSA-N 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 206010028735 Nasal congestion Diseases 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000012124 Opti-MEM Substances 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000002033 PVDF binder Substances 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 108700020479 Pancreatic hormone Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- DPWPWRLQFGFJFI-UHFFFAOYSA-N Pargyline Chemical compound C#CCN(C)CC1=CC=CC=C1 DPWPWRLQFGFJFI-UHFFFAOYSA-N 0.000 description 1
- 229920002873 Polyethylenimine Polymers 0.000 description 1
- 102000003923 Protein Kinase C Human genes 0.000 description 1
- 108090000315 Protein Kinase C Proteins 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 108090000244 Rat Proteins Proteins 0.000 description 1
- 230000010799 Receptor Interactions Effects 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 229910005948 SO2Cl Inorganic materials 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 101100043635 Solanum tuberosum SS2 gene Proteins 0.000 description 1
- 208000027520 Somatoform disease Diseases 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 238000010161 Student-Newman-Keuls test Methods 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- GCBCWTWQAFLKJG-UHFFFAOYSA-N [3-(4-chlorophenyl)-1-methoxy-1-oxopropan-2-yl]azanium;chloride Chemical compound [Cl-].COC(=O)C([NH3+])CC1=CC=C(Cl)C=C1 GCBCWTWQAFLKJG-UHFFFAOYSA-N 0.000 description 1
- 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 description 1
- ZHAFUINZIZIXFC-UHFFFAOYSA-N [9-(dimethylamino)-10-methylbenzo[a]phenoxazin-5-ylidene]azanium;chloride Chemical compound [Cl-].O1C2=CC(=[NH2+])C3=CC=CC=C3C2=NC2=C1C=C(N(C)C)C(C)=C2 ZHAFUINZIZIXFC-UHFFFAOYSA-N 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 230000002547 anomalous effect Effects 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 210000003740 anterior thalamic nuclei Anatomy 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 239000002948 appetite stimulant Substances 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 1
- 238000013320 baculovirus expression vector system Methods 0.000 description 1
- 238000010923 batch production Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229910000085 borane Inorganic materials 0.000 description 1
- UWTDFICHZKXYAC-UHFFFAOYSA-N boron;oxolane Chemical compound [B].C1CCOC1 UWTDFICHZKXYAC-UHFFFAOYSA-N 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000012592 cell culture supplement Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- MIJURWGAJOJXOR-RFJGZHNSSA-N chembl299295 Chemical compound C([C@@H]1CC[C@@H](CNS(=O)(=O)C=2C3=CC=CC=C3C=CC=2)CC1)NC(CO)CC1=CC=C(Cl)C=C1 MIJURWGAJOJXOR-RFJGZHNSSA-N 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 1
- 229960003677 chloroquine Drugs 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 229940125773 compound 10 Drugs 0.000 description 1
- 229940125782 compound 2 Drugs 0.000 description 1
- 229940125851 compound 27 Drugs 0.000 description 1
- 229940125898 compound 5 Drugs 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 239000013058 crude material Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 125000004210 cyclohexylmethyl group Chemical group [H]C([H])(*)C1([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 210000001947 dentate gyrus Anatomy 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000000586 desensitisation Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000014632 disordered eating Nutrition 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 229960003638 dopamine Drugs 0.000 description 1
- 210000002108 dorsomedial hypothalamic nucleus Anatomy 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 238000009510 drug design Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000020595 eating behavior Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 206010015037 epilepsy Diseases 0.000 description 1
- 208000028329 epileptic seizure Diseases 0.000 description 1
- 150000002148 esters Chemical group 0.000 description 1
- LJWKFGGDMBPPAZ-UHFFFAOYSA-N ethoxyethane;toluene Chemical compound CCOCC.CC1=CC=CC=C1 LJWKFGGDMBPPAZ-UHFFFAOYSA-N 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000003818 flash chromatography Methods 0.000 description 1
- 238000001506 fluorescence spectroscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000003269 fluorescent indicator Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 239000007999 glycylglycine buffer Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 210000004326 gyrus cinguli Anatomy 0.000 description 1
- 210000001753 habenula Anatomy 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
- 239000000938 histamine H1 antagonist Substances 0.000 description 1
- 239000003485 histamine H2 receptor antagonist Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 210000004283 incisor Anatomy 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 239000012194 insect media Substances 0.000 description 1
- 210000002946 intralaminar thalamic nuclei Anatomy 0.000 description 1
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 1
- FPCCSQOGAWCVBH-UHFFFAOYSA-N ketanserin Chemical compound C1=CC(F)=CC=C1C(=O)C1CCN(CCN2C(C3=CC=CC=C3NC2=O)=O)CC1 FPCCSQOGAWCVBH-UHFFFAOYSA-N 0.000 description 1
- 210000003796 lateral hypothalamic area Anatomy 0.000 description 1
- 210000003140 lateral ventricle Anatomy 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000012280 lithium aluminium hydride Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000000627 locus coeruleus Anatomy 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 229960000582 mepyramine Drugs 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- XMJHPCRAQCTCFT-UHFFFAOYSA-N methyl chloroformate Chemical compound COC(Cl)=O XMJHPCRAQCTCFT-UHFFFAOYSA-N 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 229960003955 mianserin Drugs 0.000 description 1
- 238000004452 microanalysis Methods 0.000 description 1
- 210000000218 midline thalamic nuclei Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 108091005601 modified peptides Proteins 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- RAWXKZPWRXQRSI-UHFFFAOYSA-N n-[6-[(4-aminoquinazolin-2-yl)amino]hexyl]naphthalene-1-sulfonamide Chemical compound C1=CC=C2C(N)=NC(NCCCCCCNS(=O)(=O)C=3C4=CC=CC=C4C=CC=3)=NC2=C1 RAWXKZPWRXQRSI-UHFFFAOYSA-N 0.000 description 1
- NVSYANRBXPURRQ-UHFFFAOYSA-N naphthalen-1-ylmethanamine Chemical compound C1=CC=C2C(CN)=CC=CC2=C1 NVSYANRBXPURRQ-UHFFFAOYSA-N 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 1
- KVBGVZZKJNLNJU-UHFFFAOYSA-N naphthalene-2-sulfonic acid Chemical compound C1=CC=CC2=CC(S(=O)(=O)O)=CC=C21 KVBGVZZKJNLNJU-UHFFFAOYSA-N 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000002660 neuropeptide Y receptor antagonist Substances 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 108091027963 non-coding RNA Proteins 0.000 description 1
- 102000042567 non-coding RNA Human genes 0.000 description 1
- QQOBHTXYNMYKOL-DSXOWAILSA-N npy3-36 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 QQOBHTXYNMYKOL-DSXOWAILSA-N 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- MUMZUERVLWJKNR-UHFFFAOYSA-N oxoplatinum Chemical compound [Pt]=O MUMZUERVLWJKNR-UHFFFAOYSA-N 0.000 description 1
- BHAAPTBBJKJZER-UHFFFAOYSA-N p-anisidine Chemical compound COC1=CC=C(N)C=C1 BHAAPTBBJKJZER-UHFFFAOYSA-N 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 208000027753 pain disease Diseases 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 229960001779 pargyline Drugs 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 235000021401 pellet diet Nutrition 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- MRBDMNSDAVCSSF-UHFFFAOYSA-N phentolamine Chemical compound C1=CC(C)=CC=C1N(C=1C=C(O)C=CC=1)CC1=NCCN1 MRBDMNSDAVCSSF-UHFFFAOYSA-N 0.000 description 1
- 229960001999 phentolamine Drugs 0.000 description 1
- XHXFXVLFKHQFAL-UHFFFAOYSA-N phosphoryl trichloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229910003446 platinum oxide Inorganic materials 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 229920006316 polyvinylpyrrolidine Polymers 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000012746 preparative thin layer chromatography Methods 0.000 description 1
- CMDGQTVYVAKDNA-UHFFFAOYSA-N propane-1,2,3-triol;hydrate Chemical compound O.OCC(O)CO CMDGQTVYVAKDNA-UHFFFAOYSA-N 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- BLGXFZZNTVWLAY-DIRVCLHFSA-N rauwolscine Chemical compound C1=CC=C2C(CCN3C[C@H]4CC[C@H](O)[C@H]([C@H]4C[C@H]33)C(=O)OC)=C3NC2=C1 BLGXFZZNTVWLAY-DIRVCLHFSA-N 0.000 description 1
- 238000001525 receptor binding assay Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009712 regulation of translation Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 239000003772 serotonin uptake inhibitor Substances 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- DKGZKTPJOSAWFA-UHFFFAOYSA-N spiperone Chemical compound C1=CC(F)=CC=C1C(=O)CCCN1CCC2(C(NCN2C=2C=CC=CC=2)=O)CC1 DKGZKTPJOSAWFA-UHFFFAOYSA-N 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000005556 structure-activity relationship Methods 0.000 description 1
- 229940124530 sulfonamide Drugs 0.000 description 1
- 150000003456 sulfonamides Chemical class 0.000 description 1
- 150000003461 sulfonyl halides Chemical class 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 210000000221 suprachiasmatic nucleus Anatomy 0.000 description 1
- 210000004377 supraoptic nucleus Anatomy 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 229960000278 theophylline Drugs 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-M toluene-4-sulfonate Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-M 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000003151 transfection method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- LEIMLDGFXIOXMT-UHFFFAOYSA-N trimethylsilyl cyanide Chemical compound C[Si](C)(C)C#N LEIMLDGFXIOXMT-UHFFFAOYSA-N 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000003828 vacuum filtration Methods 0.000 description 1
- 210000004440 vestibular nuclei Anatomy 0.000 description 1
- 238000004260 weight control Methods 0.000 description 1
- 238000011684 zucker rat (obese) Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Neuropeptide Y is a member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY and its relatives (peptide YY or PYY, and pancreatic polypeptide or PP) elicit a broad range of physiological effects through activation of at least five G protein-coupled receptor subtypes known as Y1, Y2, Y3, Y4 (or PP), and the "atypical Y1". The role of NPY as the most powerful stimulant of feeding behavior yet described is thought to occur primarily through activation of the hypothalamic "atypical Y1" receptor.
- This receptor is unique in that its classification was based solely on feeding behavior data, rather than radioligand binding data, unlike the Y1, Y2, Y3, and Y4 (or PP) receptors, each of which were described previously in both radioligand binding and functional assays.
- NPY The peptide neurotransmitter neuropeptide Y
- the peptide neurotransmitter neuropeptide Y is a 36 amino acid member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY is considered to be the most powerful stimulant of feeding behavior yet described (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Direct injection into the hypothalamus of satiated rats, for example, can increase food intake up to 10-fold over a 4-hour period (Stanley et al., 1992).
- Rank orders of affinity for key peptides are based on previously reported binding and functional data (Schwartz et al., 1990; Wahlestedt et al., 1991; Dumont et al., 1992; Wahlestedt and Reis, 1993).
- Data for the Y2 receptor were disclosed in PCT International Application No. PCT/US95/01469, filed February 3, 1995, International Publication No. WO 95/21245, published August 10, 1995 the foregoing contents of which are hereby incorporated by reference.
- Data for the Y4 receptor were disclosed in PCT International Application No.
- NPY receptor pharmacology has historically been based on structure/activity relationships within the pancreatic polypeptide family.
- the entire family includes the namesake pancreatic polypeptide (PP), synthesized primarily by endocrine cells in the pancreas; peptide YY (PYY), synthesized primarily by endocrine cells in the gut; and NPY, synthesized primarily in neurons (Michel, 1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). All pancreatic polypeptide family members share a compact structure involving a "PP-fold" and a conserved C- terminal hexapeptide ending in Tyr 36 (or Y 36 in the single letter code).
- Y-type receptors The striking conservation of Y 36 has prompted the reference to the pancreatic polypeptides' receptors as "Y-type" receptors (Wahlestedt et al., 1987), all of which are proposed to function as seven transmembrane-spanning G protein-coupled receptors (Dumont et al., 1992).
- the Y1 receptor recognizes NPY ⁇ PYY >> PP (Gêtmar et al., 1992).
- the receptor requires both the N- and the C- terminal regions of the peptides for optimal recognition. Exchange of Gln 34 in NPY or PYY with the analogous residue from PP (Pro 34 ), however, is well-tolerated.
- the Y1 receptor has been cloned from a variety of species including human, rat and mouse (Larhammar et al, 1992; Herzog et al, 1992; Eva et al, 1990; Eva et al, 1992).
- the Y2 receptor recognizes PYY ⁇ NPY PP and is relatively tolerant of N-terminal deletion (Gêtmar et al., 1992).
- the receptor has a strict requirement for structure in the C-terminus (Arg 33 -Gln 34 -Arg 35 -Tyr 36 -NH 2 ); exchange of Gln 34 with Pro 34 , as in PP, is not well tolerated.
- the Y2 receptor has recently been cloned.
- the Y3 receptor is characterized by a strong preference for NPY over PYY and PP (Wahlestedt et al., 1991). [Pro 34 ]NPY is reasonably well tolerated even though PP, which also contains Pro 34 , does not bind well to the Y3 receptor.
- the Y3 receptor (Y3) has not yet been cloned.
- the Y4 receptor binds PP > PYY > NPY. Like the Y1, the Y4 requires both the N- and the C-terminal regions of the peptides for optimal recognition.
- the "atypical Y1" or “feeding" receptor was defined exclusively by injection of several pancreatic polypeptide analogs into the paraventricular nucleus of the rat hypothalamus which stimulated feeding behavior with the following rank order: NPY 2-36 ⁇ NPY ⁇ PYY ⁇ [Leu 31 , Pro 34 ] NPY > NPY 13-36 (Kalra et al., 1991; Stanley et al., 1992). The profile is similar to that of a Y1-like receptor except for the anomalous ability of NPY 2-36 to stimulate food intake with potency equivalent or better than that of NPY. A subsequent report in J. Med . Chem . by Balasubramaniam et al.
- This invention now reports the isolation by expression cloning of a novel Y-type receptor from a rat hypothalamic cDNA library, along with its pharmacological characterization, in situ localization, and human homolog.
- the data provided link this newly-cloned receptor subtype, from now on referred to as the Y5 subtype, to the "atypical Y1" feeding response.
- This discovery therefore provides a novel approach, through the use of heterologous expression systems, to develop a subtype selective antagonist for obesity and other indications.
- This invention is based on the use of a 125 I-PYY-based expression cloning technique to isolate a rat hypothalamic cDNA encoding an "atypical Y1" receptor referred to herein as the Y5 receptor subtype.
- This application also concerns the isolation and characterization of a Y5 homolog from human hippocampus. Protein sequence analysis reveals that the Y5 receptor belongs to the G protein- coupled receptor superfamily. Both the human and rat homolog display ⁇ 42% identity in transmembrane domains with the previously cloned "Y-type" receptors. Rat brain localization studies using in situ hybridization techniques verified the existence of Y5 receptor mRNA in rat hypothalamus.
- disorders or diseases associated with the inhibition of the Y5 receptor subtype especially diseases caused by eating disorders like obesity, bulimia nervosa, diabetes, dislipidimia
- diseases caused by eating disorders like obesity, bulimia nervosa, diabetes, dislipidimia may be effected by administration of compounds which bind selectively to the Y5 receptor and inhibit the activation of the Y5 receptor.
- any disease states in which the Y5 receptor subtype is involved for example, memory loss, epileptic seizures, migraine, sleep disturbance, pain, and affective disorders such as depression and anxiety may also be treated using compounds which bind selectively to the Y5 receptor.
- This invention provides a method of modifying a subject's feeding behavior which comprises administering to the subject a compound which is a Y5 receptor agonist or antagonist in an amount effective to alter the subject's consumption of food and thereby modify the subject's feeding behavior.
- This invention also provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the binding of the compound to the human receptor is characterized by a K i less than 100 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount.
- this invention provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the compound's binding to the human Y5 receptor is characterized by a K i less than 10 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount.
- This invention further provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 100 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 1000 nanomolar when measured in the presence of 125 I- PYY in a predetermined amount.
- This invention also provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 1 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount; and (b) the compound's binding to any other human Y-type receptor is characterized by a K i greater than 100 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount.
- This invention further provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 1 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 25 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount.
- This invention provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 0.1 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 1 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount.
- This invention further provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 0.01 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount ; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 1 nanomolar when measured in the presence of 125 I-PYY in a predetermined amount.
- this invention provides an isolated nucleic acid encoding a Y5 receptor.
- This invention also provides an isolated Y5 receptor protein.
- This invention provides a vector comprising the above-described nucleic acid.
- This invention also provides a plasmid which comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to the DNA encoding the human Y5 receptor as to permit expression thereof designated pcEXV-hY5 (ATCC Accession No. 75943).
- This invention further provides a plasmid which comprises the regulatory elements necessary for expression of DNA in a mammalian cell operatively linked to the DNA encoding the rat Y5 receptor as to permit expression thereof designated pcEXV-rY5 (ATCC Accession No. 75944).
- This invention provides a mammalian cell comprising the above-described plasmid or vector.
- This invention also provides a nucleic acid probe comprising a nucleic acid of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid encoding a Y5 receptor.
- this invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding a Y5 receptor so as to prevent translation of the mRNA.
- This invention also provides an antibody directed to a Y5 receptor.
- This invention provides a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce activity of a human Y5 receptor by passing through a cell membrane and binding specifically with mRNA encoding a human Y5 receptor in the cell so as to prevent its translation and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
- This invention also provides a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of a human Y5 receptor and a pharmaceutically acceptable carrier.
- This invention further provides a pharmaceutical composition comprising an amount of an agonist effective to increase activity of a Y5 receptor and a pharmaceutically acceptable carrier.
- This invention further provides the above-described pharmaceutical composition which comprises an amount of an antibody effective to block binding of a ligand to the Y5 receptor and a pharmaceutically acceptable carrier.
- This invention additionally provides a transgenic nonhuman mammal expressing DNA encoding a human Y5 receptor.
- This invention also provides a method for determining whether a ligand can specifically bind to a Y5 receptor which comprises contacting a plurality of cells transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the ligand under conditions permitting binding of ligands to such receptor, detecting the presence of any such ligand specifically bound to the Y5 receptor, and thereby determining whether the ligand specifically binds to the Y5 receptor.
- This invention further provides a method for determining whether a ligand is a Y5 receptor agonist which comprises contacting a cell transfected with and expressing nucleic acid encoding a human Y5 receptor with the ligand under conditions permitting activation of the Y5 receptor, detecting an increase in Y5 receptor activity, and thereby determining whether the ligand is a human Y5 receptor agonist.
- This invention provides a method for determining whether a ligand is a Y5 receptor antagonist which comprises contacting a cell transfected with and expressing DNA encoding a Y5 receptor with the ligand in the presence of a known Y5 receptor agonist, such as PYY or NPY, under conditions permitting the activation of the Y5 receptor, detecting a decrease in Y5 receptor activity, and thereby determining whether the ligand is a Y5 receptor antagonist.
- a known Y5 receptor agonist such as PYY or NPY
- This invention further provides a method of screening a plurality of chemical compounds not known to bind to a Y5 receptor to identify a compound which specifically binds to the Y5 receptor, which comprises (a) contacting a cell transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction from a cell extract of such cells, with a compound known to bind specifically to the Y5 receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the Y5 receptor, under conditions permitting binding of compounds known to bind the Y5 receptor; (c) determining whether the binding of the compound known to bind to the Y5 receptor is reduced in the presence of the compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the Y5 receptor of each compound included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the Y5 receptor.
- This invention also provides a method of screening a plurality of chemical compounds not known to activate a Y5 receptor to identify a compound which activates the Y5 receptor which comprises (a) contacting a cell transfected with and expressing the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the plurality of compounds not known to bind specifically to the Y5 receptor, under conditions permitting activation of the Y5 receptor; (b) determining whether the activity of the Y5 receptor is increased in the presence of the compounds; and if so (c) separately determining whether the activation of the Y5 receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the Y5 receptor.
- This invention further provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a Y5 receptor to identify a compound which inhibits the activation of the Y5 receptor, which comprises (a) contacting a cell transfected with and expressing the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the plurality of compounds in the presence of a known Y5 receptor agonist, under conditions permitting activation of the Y5 receptor; (b) determining whether the activation of the Y5 receptor is reduced in the presence of the plurality of compounds, relative to the activation of the Y5 receptor in the absence of the plurality of compounds; and if so (c) separately determining the inhibition of activation of the Y5 receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the Y5 receptor.
- this invention provides a process for identifying a chemical compound which specifically binds to a Y5 receptor, which comprises contacting nonneuronal cells expressing on their cell surface the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the chemical compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the Y5 receptor.
- This invention also provides a process involving competitive binding for identifying a chemical compound which specifically binds to a Y5 receptor which comprises separately contacting nonneuronal cells expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the Y5 receptor, a decrease in the binding of the second chemical compound to the Y5 receptor in the presence of the chemical compound indicating that the chemical compound binds to the Y5 receptor.
- This invention further provides a process for determining whether a chemical compound specifically binds to and activates a Y5 receptor, which comprises contacting nonneuronal cells producing a second messenger response and expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with the chemical compound under conditions suitable for activation of the Y5 receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in second messenger response in the presence of the chemical compound indicating that the chemical compound activates the Y5 receptor.
- This invention also provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a Y5 receptor, which comprises separately contacting nonneuronal cells producing a second messenger response and expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with both the chemical compound and a second chemical compound known to activate the Y5 receptor, and with only the second chemical compound, under conditions suitable for activation of the Y5 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in second messenger response in the presence of both the chemical compound and the second chemical compound indicating that the chemical compound inhibits activation of the Y5 receptor.
- This invention additionally provides a method of treating a subject's abnormality, wherein the abnormality is alleviated by the inhibition of a Y5 receptor which comprises administering to a subject an effective amount of Y5 receptor antagonist.
- This invention also provides a method of treating a subject's abnormality wherein the abnormality is alleviated by the activation of a Y5 receptor which comprises administering to a subject an effective amount of a Y5 receptor agonist.
- This invention further provides a method for diagnosing a predisposition to a disorder associated with the activity of a specific allelic form of a human Y5 receptor which comprises: a. obtaining DNA from a subject to be tested; digesting the DNA with restriction enzymes; c. separating the resulting DNA fragments; d. contacting the fragments with a detectably labeled nucleic acid probe capable of specifically hybridizing with a sequence uniquely present within the sequence of a nucleic acid molecule encoding the allelic form of the human Y5 receptor; and e. detecting the presence of labeled probe from the subject to be tested, the presence of such hybridized probe indicating that the subject is predisposed to the disorder.
- This invention also provides a method of preparing the isolated Y5 receptor which comprises: a. inserting nucleic acid encoding Y5 receptor in a suitable vector which comprises the regulatory elements necessary for expression of the nucleic acid operatively linked to the nucleic acid encoding a Y5 receptor; b. inserting the resulting vector in a suitable host cell so as to obtain a cell which produces the Y5 receptor; c. recovering the receptor produced by the resulting cell; and d. purifying the receptor so recovered.
- FIG. 1 Competitive displacement of 125 I-PYY on membranes from rat hypothalamus. Membranes were incubated with 125 I- PYY and increasing concentrations of peptide competitors. IC 50 values corresponding to 50% displacement were determined by nonlinear regression analysis. Data are representative of at least two independent experiments. IC 50 values for these compounds are listed separately in Table 2.
- FIG. 1 Competitive displacement of 125 I-PYY 3-36 on membranes from rat hypothalamus. Membranes were incubated with 125 I-PYY 3-36 and increasing concentrations of peptide competitors. IC 50 values corresponding to 50% displacement were determined by nonlinear regression analysis. Data are representative of at least two independent experiments. IC 50 values for these compounds are listed separately in Table 2.
- FIG. 3 Nucleotide sequence of the rat hypothalamic Y5 cDNA clone (Seq. I.D. No 1). Initiation and stop codons are underlined. Only partial 5' and 3' untranslated sequences are shown.
- Figure 4 Corresponding amino acid sequence of the rat hypothalamic Y5 cDNA clone (Seq. I.D. No. 2).
- FIG. 5 Nucleotide sequence of the human hippocampal Y5 cDNA clone (Seq. I.D. No. 3). Initiation and stop codons are underlined. Only partial 5' and 3' untranslated sequences are shown.
- Figure 6 Corresponding amino acid sequence of the human hippocampal Y5 cDNA clone(Seq. I.D. No. 4).
- FIG. 7 A-E Comparison of coding nucleotide sequences between rat hypothalamic Y5 (top row) and human hippocampal Y5 (bottom row) cDNA clones (84.1% nucleotide identity).
- F-G Comparison of deduced amino acid sequences between rat hypothalamic Y5 (top row) and human hippocampal Y5 (bottom row) cDNA clones (87.2% overall and 98.8% transmembrane domain identities).
- FIG. 8 Comparison of the human Y5 receptor deduced amino acid sequence with those of the human Y1, Y2, Y4 sequences. Solid bars, the seven putative membrane- spanning domains (TM I-VII). Shading, identities between receptor sequences.
- FIG. 11 Competitive displacement of 125 I-PYY from COS-7 cells transiently expressing rat Y5 receptors.
- Membranes were incubated with 125 I-PYY and increasing concentrations of peptide competitors.
- Data are representative of at least two independent experiments. Rank orders of affinity for these and other compounds are listed separately in Table 4.
- Figure 12 Inhibition of forskolin-stimulated cAMP accumulation in intact 293 cells stably expressing rat Y5 receptors.
- Functional data were derived from radioimmunoassay of cAMP in 293 cells stimulated with 10 ⁇ M forskolin over a 5 minute period.
- Rat/human NPY was tested for agonist activity at concentrations ranging from 0.03 pM to 0.3 ⁇ M over the same period.
- the EC 50 value corresponding to 50% maximal activity was determined by nonlinear regression analysis. The data shown are representative of three independent experiments.
- FIG. 13 Schematic diagrams of coronal sections through the rat brain, illustrating the distribution of NPY Y5 receptor mRNA, as visualized microscopically in sections dipped in liquid emulsion.
- the sections are arranged from rostral (A) to caudal (H). Differences in silver grain density over individual neurons in a given area are indicated by the hatching gradient.
- A rostral
- H caudal
- Aco anterior cortical amygdaloid nucleus
- AD anterodorsal thalamic nucleus
- APT anterior pretectal nucleus
- Arc arcuate hypothalamic nucleus
- BLA basolateral amygdaloid nucleus anterior
- CA3 field CA3 of Ammon's horn, hippocampus
- CeA central amygdaloid nucleus
- Cg cingulate cortex
- CL centrolateral thalamic nucleus
- CM central medial thalamic nucleus
- DG dentate gyrus, hippocampus
- DMH dorsomedial hypothalamic nucleus
- GiA gigantocellular reticular nucleus, alpha
- HDB nucleus horizontal limb diagonal band
- LH lateral hypothalamic area
- MePV medial amygdaloid nucleus
- MVe medial vestibular nucleus
- MHb medial habenular nucleus
- MPN medial preoptic nucleus
- PC paracentral thalamic nucleus
- PrS presubiculum
- PN pontine nuclei
- PVH paraventricular hypothalamic nucleus
- PVHmp paraventricular hypothalamic nucleus, medial parvicellular part
- PVT paraventricular thalamic nucleus
- RLi rostral linear nucleus raphe
- RSG retrosplenial cortex
- SCN suprachiasmatic nucleus
- Figure 15 Corresponding partial amino acid sequence of the canine Y5 cDNA clone (Seq. I.D. No. 6).
- Figure 16 A. Northern blot analysis of various rat tissues.
- Figure 17 Southern blot analysis of human (A) or rat(B) genomic DNA encoding the Y5 receptor subtype.
- Figure 20 NPY-Dependent Inhibition of Forskolin Stimulated cAMP Accumulation by Cloned Y5 Receptors. Intact cells stably transfected with human or rat Y5 receptors were incubated with forskolin plus a range of human NPY concentrations as indicated. A representative experiment is shown for each receptor system (n ⁇ 2).
- FIG. 21 Calcium Mobilization: Fura-2 Assay. Cloned human Y-type receptors in the host cells indicated were screened for intracellular calcium mobilization in response to NPY and related peptides. Representative calcium transients are shown for each receptor system.
- Figure 23 Nucleotide sequence of the canine Y5 cDNA clone (Seq. I.D. No. 13). Initiation and stop codons are underlined. Only partial 5' and 3' untranslated sequences are shown.
- Figure 24 Corresponding amino acid sequence of the canine Y5 cDNA clone (Seq. I.D. No. 14).
- FIG 25 Schematic representation of the human Y1/Y5 locus on chromosome 4q. Open boxes represent non-coding exons. Closed boxes indicate coding regions (CDS). Arrows on top of exons 1A, 1B and 1C show transcription starts for the three known alternative splice variants of the Y1 mRNA (Ball, et al., 1995). Arrows under the coding regions show opposite transcriptional directions for the Y1 and Y5 genes. "p*" indicates a PstI restriction site polymorphism described previously in the Y1 locus (Herzog, et aI., 1993).
- agonist is used throughout this application to indicate any peptide or non-peptidyl compound which increases the activity of any of the receptors of the subject invention.
- antagonist is used throughout this application to indicate any peptide or non-peptidyl compound which decreases or inhibits the activity of any of the receptors of the subject invention.
- the activity of a G-protein coupled receptor such as a Y5 receptor may be measured using any of a variety of appropriate functional assays in which activation of the receptor in question results in an observable change in the level of some second messenger system, including but not limited to adenylate cyclase, calcium mobilization, inositol phospholipid hydrolysis or guanylyl cyclase.
- This invention provides a method of modifying a subject's feeding behavior which comprises administering to the subject a compound which is a Y5 receptor agonist or antagonist in an amount effective to alter the subject's consumption of food and thereby modify the subject's feeding behavior.
- the compound is a Y5 receptor antagonist and the amount is effective to decrease the consumption of food by the subject.
- the compound is administered in combination with food.
- the compound is a Y5 receptor agonist and the amount is effective to increase the consumption of food by the subject.
- the compound is administered in combination with food.
- the subject may be a vertebrate, a mammal, a human or a canine subject.
- This invention also provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the binding of the compound to the human Y5 receptor is characterized by a K i less than 100 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the compound has a K i less than 50 nanomolar.
- the compound has a K i less than 10 nanomolar.
- the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 10 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 50 nanomolar.
- the binding of the compound is characterized by a K i greater than 100 nanomolar.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to each of the human Y1, human Y2 and human Y4 receptors.
- the feeding disorder may be obesity or bulimia.
- the subject may be a vertebrate, a mammal, a human or a canine subject.
- This invention further provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the compound's binding to the human Y5 receptor is characterized by a K i less than 10 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the compound's binding is characterized by a K i less than 1 nanomolar.
- the compound's binding to any other human Y-type receptor is characterized by a K i greater than 10 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the compound's binding to each of the human Y1, human Y2, and human Y4 receptors is characterized by a K i greater than 10 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the compound's binding to any other human Y-type receptor is characterized by a K i greater than 50 nanomolar.
- the compound's binding to any other human Y-type receptor is characterized by a K i greater than 100 nanomolar.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to each of the human Y1, human Y2, and human Y4 receptors.
- the feeding disorder may be obesity or bulimia.
- the subject may be a vertebrate, a mammal, a human, or a canine subject.
- This invention provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 100 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 1000 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the binding of the compound to the human Y5 receptor is characterized by a K i less than 10 nanomolar.
- This invention also provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 1 nanomolar when measured in the presence in 125 I-PYY; and (b) the compound's binding to any other human Y-type receptor is characterized by a K i greater than 100 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor. In another embodiment, the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to each of the human Y1, human Y2, and human Y4 receptors.
- the feeding disorder may be anorexia.
- the subject may be a vertebrate, a mammal, a human, or a canine subject.
- This invention further provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 1 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 25 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- This invention provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 0.1 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 1 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration. In one embodiment, the binding of the agonist to any other human Y-type receptor is characterized by a K i greater than 10 nanomolar.
- This invention provides a method of treating a subject's feeding disorder which comprises administering to the subject a peptidyl compound which is a Y5 receptor agonist in an amount effective to increase the activity of the subject's Y5 receptor, wherein (a) the binding of the compound to the human Y5 receptor is characterized by a K i less than 0.01 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration; and (b) the binding of the compound to any other human Y-type receptor is characterized by a K i greater than 1 nanomolar when measured in the presence of 125 l-PYY at a predetermined concentration.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor. In another embodiment, the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to each of the human Y1, human Y2, and human Y4 receptors.
- the feeding disorder is anorexia.
- the subject may be a vertebrate, a mammal, a human, or a canine subject.
- this invention provides an isolated nucleic acid encoding a Y5 receptor.
- the Y5 receptor is a vertebrate or a mammalian Y5 receptor.
- the Y5 receptor is a human Y5 receptor.
- the isolated nucleic acid encodes a receptor being characterized by an amino acid sequence in the transmembrane region, wherein the amino acid sequence has 60% homology or higher to the amino acid sequence in the transmembrane region of the human Y5 receptor shown in Figure 6.
- the Y5 receptor has substantially the same amino acid sequence as described in Figure 4.
- the Y5 receptor has substantially the same amino acid sequence as described in Figure 6.
- the Y5 receptor has substantially the same amino acid sequence as described in Figure 24.
- the nucleic acid is a DNA.
- the DNA is a cDNA.
- the DNA is a genomic DNA.
- the nucleic acid is RNA.
- the nucleic acid encodes a human Y5 receptor.
- the human Y5 receptor has the amino acid sequence as described in Figure 6.
- the nucleic acid encodes a rat Y5 receptor.
- the rat Y5 receptor has the amino acid sequence as shown in Figure 4.
- the nucleic acid encodes a canine Y5 receptor.
- the canine Y5 receptor has the amino acid sequence shown in Figure 24.
- This invention further provides DNA which is degenerate with any of the DNA shown in Figures 3, 5, 14 and 23, wherein the DNA encodes Y5 receptors having the amino acid sequences shown in Figures 4, 6, 15 and 24, respectively.
- This invention also encompasses DNAs and cDNAs which encode amino acid sequences which differ from those of Y5 receptor, but which should not produce phenotypic changes.
- this invention also encompasses DNAs and cDNAs which hybridize to the DNA, RNA, and cDNA of the subject invention. Hybridization methods are well known to those of skill in the art.
- the nucleic acid of the subject invention also includes nucleic acid coding for polypeptide analogs, fragments or derivatives of antigenic polypeptides which differ from naturally-occurring forms in terms of the identity or location of one or more amino acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues and addition analogs where in one or more amino acid residues is added to a terminal or medial portion of the polypeptides) and which share some or all properties of naturally-occurring forms.
- nucleic acids include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate nucleic acid sequences that facilitate construction of readily expressed vectors.
- nucleic acids described and claimed herein are useful for the information which they provide concerning the amino acid sequence of the polypeptide and as products for the large scale synthesis of the polypeptide by a variety of recombinant techniques.
- the nucleic acid is useful for generating new cloning and expression vectors, transformed and transfected prokaryotic and eukaryotic host cells, and new and useful methods for cultured growth of such host cells capable of expression of the polypeptide and related products.
- the nucleic acid encodes a rat Y5 receptor.
- the rat Y5 receptor has the amino acid sequence shown in Figure 4.
- This invention also provides an isolated Y5 receptor protein.
- the Y5 receptor protein is a human Y5 receptor protein.
- the human Y5 receptor protein has the amino acid sequence as shown in Figure 6.
- the Y5 receptor protein is a rat Y5 receptor protein.
- the rat Y5 receptor protein has the amino acid sequence as shown in Figure 4.
- the Y5 receptor protein is a canine Y5 receptor protein.
- the canine Y5 receptor protein has the amino acid sequence as shown in Figure 24.
- This invention provides a vector comprising the above- described nucleic acid.
- Vectors which comprise the isolated nucleic acid described hereinabove also are provided.
- Suitable vectors comprise, but are not limited to, a plasmid or a virus. These vectors may be transformed into a suitable host cell to form a host cell vector system for the production of a polypeptide having the biological activity of a Y5 receptor.
- This invention provides the above-described vector adapted for expression in a cell which further comprises the regulatory elements necessary for expression of the nucleic acid in the cell operatively linked to the nucleic acid encoding the Y5 receptor as to permit expression thereof.
- the cell is a Xenopus cell such as an oocyte or melanophore.
- This invention provides the above-described vector adapted for expression in a bacterial cell which further comprises the regulatory elements necessary for expression of the nucleic acid in the bacterial cell operatively linked to the nucleic acid encoding the Y5 receptor as to permit expression thereof.
- This invention provides the above-described vector adapted for expression in a yeast cell which comprises the regulatory elements necessary for expression of the nucleic acid in the yeast cell operatively linked to the nucleic acid encoding the Y5 receptor as to permit expression thereof.
- This invention provides the above-described vector adapted for expression in an insect cell which comprises the regulatory elements necessary for expression of the nucleic acid in the insect cell operatively linked to the nucleic acid encoding the Y5 receptor as to permit expression thereof.
- the vector is adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the mammalian cell operatively linked to the nucleic acid encoding the mammalian Y5 receptor as to permit expression thereof.
- the vector is adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the nucleica acid in the mammalian cell operatively linked to the nucleic acid encoding the canine Y5 receptor as to permit expression thereof.
- the vector is adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the mammalian cell operatively linked to the nucleic acid encoding the human Y5 receptor as to permit expression thereof.
- the plasmid is adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the mammalian cell operatively linked to the nucleic acid encoding the rat Y5 receptor as to permit expression thereof.
- the plasmid is adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of the nucleic acid in the mammalian cell operatively linked to the nucleic acid encoding the canine Y5 receptor as to permit expression thereof.
- This invention provides the above-described plasmid adapted for expression in a mammalian cell which comprises the regulatory elements necessary for expression of nucleic acid in a mammalian cell operatively linked to the nucleic acid encoding the mammalian Y5 receptor as to permit expression thereof.
- This invention provides a plasmid which comprises the regulatory elements necessary for expression of nucleic acid in a mammalian cell operatively linked to the nucleic acid encoding the human Y5 receptor as to permit expression thereof designated pcEXV-hY5 (ATCC Accession No. 75943).
- This plasmid (pcEXV-hY5) was deposited on November 4, 1994 with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Accession No. 75943.
- ATCC American Type Culture Collection
- This invention provides a plasmid which comprises the regulatory elements necessary for expression of nucleic acid in a mammalian cell operatively linked to the nucleic acid encoding the rat Y5 receptor as to permit expression thereof designated pcEXV-rY5 (ATCC Accession No. 75944).
- This plasmid (pcEXV-rY5) was deposited on November 4, 1994 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Accession No. CRL 75944.
- This invention provides a plasmid designated Y5-bd-5 (ATCC Accession No. 97355).
- This invention also provides a plasmid designated Y5-bd-8 (ATCC Accession No. 97354). These plasmids were deposited on December 1, 1995 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure .
- This invention further provides a plasmid designated c55- BO11, which comprises a canine Y5 receptor. This plasmid was deposited on May 29, 1996 with the ATCC under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganism for the Purposes of Patent procedure, and was accorded ATCC Accession No. 97587.
- This invention provides a baculovirus designated hY5-BB3 (ATCC Accession No. VR-2520).
- This baculovirus was deposited on November 15, 1995 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Accession No. VR- 2520.
- This invention provides a mammalian cell comprising the above-described plasmid or vector.
- the mammalian cell is a COS-7 cell, a Chinese hamster ovary (CHO) cell, or a neuronal cell such as the glial cell line C6.
- the mammalian cell is a 293 human embryonic kidney cell designated 293-rY5-14 (ATCC Accession No. CRL 11757).
- This cell (293-rY5-14) was deposited on November 4, 1994 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and was accorded ATCC Accession No. CRL 11757.
- the mammalian cell is a mouse fibroblast LM(tk-) cell, containing the plasmid pcEXV-hY5 and designated L-hY5-7 (ATCC Accession No. CRL-11995).
- the mammalian cell is a mouse embryonic N1H-3T3 cell containing the plasmid pcEXV-hY5 and designated N-hY5-8 (ATCC Accession No. CRL-11994) .
- These cells were deposited on November 15, 1995 with the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure, and were accorded ATCC Accession Nos. CRL-11995 and CRL-11994, respectively.
- This invention provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence included within the sequence of a nucleic acid molecule encoding a Y5 receptor.
- the nucleic acid is DNA.
- This nucleic acid produced can either be DNA or RNA.
- the phrase "specifically hybridizing” means the ability of a nucleic acid to recognize a nucleic acid sequence complementary to its own and to form double- helical segments through hydrogen bonding between complementary base pairs.
- This nucleic acid of at least 15 nucleotides capable of specifically hybridizing with a sequence of a nucleic acid encoding the human Y5 receptors can be used as a probe.
- Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary greatly in length and may be labeled with a detectable label, such as a radioisotope or fluorescent dye, to facilitate detection of the probe.
- DNA probe molecules may be produced by insertion of a DNA molecule which encodes the Y5 receptor into suitable vectors, such as plasmids or bacteriophages, followed by transforming into suitable bacterial host cells, replication in the transformed bacterial host cells and harvesting of the DNA probes, using methods well known in the art.
- probes may be generated chemically from DNA synthesizers.
- RNA probes may be generated by inserting the DNA which encodes the Y5 receptor downstream of a bacteriophage promoter such as T3, T7 or SP6. Large amounts of RNA probe may be produced by incubating the labeled nucleotides with the linearized fragment where it contains an upstream promoter in the presence of the appropriate RNA polymerase.
- This invention also provides a nucleic acid of at least 15 nucleotides capable of specifically hybridizing with a sequence of a nucleic acid which is complementary to the mammalian nucleic acid encoding a Y5 receptor.
- This nucleic acid may either be a DNA or RNA molecule.
- This invention further provides a nucleic acid probe molecule of at least 15 nucleotides which is complementary to a unique fragment of the sequence of the nucleic acid molecule encoding a Y5 receptor.
- This invention also provides a nucleic acid probe comprising a nucleic acid molecule of at least 15 nucleotides which is complementary to the antisense sequence of a unique fragment of the sequence of a nucleic acid molecule encoding a Y5 receptor.
- the Y5 receptor is a mammalian receptor. In further embodiments, the Y5 receptor is a human, rat, or canine receptor.
- This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to mRNA encoding a Y5 receptor so as to prevent translation of the mRNA.
- This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the genomic DNA of a Y5 receptor.
- This invention provides an antisense oligonucleotide of a Y5 receptor comprising chemical analogues of nucleotides.
- This invention further provides an antibody directed to a Y5 receptor.
- This invention also provides an antibody directed to a human Y5 receptor.
- This invention also provides a monoclonal antibody directed to an epitope of a human Y5 receptor present on the surface of a Y5 receptor expressing cell.
- this invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an amount of the oligonucleotide effective to reduce activity of a human Y5 receptor by passing through a cell membrane and binding specifically with mRNA encoding a human Y5 receptor in the cell so as to prevent its translation and a pharmaceutically acceptable carrier capable of passing through a cell membrane.
- the oligonucleotide is coupled to a substance which inactivates mRNA.
- the substance which inactivates mRNA is a ribozyme.
- the pharmaceutically acceptable carrier capable of passing through a cell membrane comprises a structure which binds to a receptor specific for a selected cell type and is thereby taken up by cells of the selected cell type.
- This invention additionally provides a pharmaceutical composition comprising an amount of an antagonist effective to reduce the activity of a human Y5 receptor and a pharmaceutically acceptable carrier.
- This invention also provides a pharmaceutical composition comprising an amount of an agonist effective to increase activity of a Y5 receptor and a pharmaceutically acceptable carrier.
- This invention further provides a pharmaceutical composition comprising and effective amount of a chemical compound identified by the above- described methods and a pharmaceutically acceptable carrier.
- This invention also provides the above- described pharmaceutical composition which comprises an amount of the antibody effective to block binding of a ligand to the Y5 receptor and a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carriers means any of the standard pharmaceutically acceptable carriers. Examples include, but are not limited to, phosphate buffered saline, physiological saline, water and emulsions, such as oil/water emulsions.
- This invention provides a transgenic nonhuraan mammal expressing DNA encoding a human Y5 receptor.
- This invention provides a transgenic nonhuman mammal comprising a homologous recombination knockout of the native Y5 receptor.
- This invention provides a transgenic nonhuman mammal whose genome comprises antisense DNA complementary to DNA encoding a human Y5 receptor so placed as to be transcribed into antisense mRNA which is complementary to mRNA encoding a Y5 receptor and which hybridizes to mRNA encoding a Y5 receptor thereby reducing its translation.
- This invention provides the above-described transgenic nonhuman mammal, wherein the DNA encoding a human Y5 receptor additionally comprises an inducible promoter.
- This invention provides the transgenic nonhuman mammal, wherein the DNA encoding a human Y5 receptor additionally comprises tissue specific regulatory elements.
- the transgenic nonhuman mammal is a mouse.
- Animal model systems which elucidate the physiological and behavioral roles of Y5 receptor are produced by creating transgenic animals in which the activity of the Y5 receptor is either increased or decreased, or the amino acid sequence of the expressed Y5 receptor is altered, by a variety of techniques.
- these techniques include, but are not limited to: 1) Insertion of normal or mutant versions of DNA encoding a Y5 receptor, by microinjection, electroporation, retroviral transfection or other means well known to those skilled in the art, into appropriate fertilized embryos in order to produce a transgenic animal or 2) Homologous recombination of mutant or normal, human or animal versions of these genes with the native gene locus in transgenic animals to alter the regulation of expression or the structure of these Y5 receptor sequences.
- homologous recombination is well known in the art. It replaces the native gene with the inserted gene and so is useful for producing an animal that cannot express native Y5 receptors but does express, for example, an inserted mutant Y5 receptor, which has replaced the native Y5 receptor in the animal's genome by recombination, resulting in underexpression of the transporter. Microinjection adds genes to the genome, but does not remove them, and so is useful for producing an animal which expresses its own and added Y5 receptors, resulting in overexpression of the Y5 receptors.
- transgenic animal One means available for producing a transgenic animal, with a mouse as an example, is as follows: Female mice are mated, and the resulting fertilized eggs are dissected out of their oviducts. The eggs are stored in an appropriate medium such as M2 medium. DNA or cDNA encoding a Y5 receptor is purified from a vector by methods well known in the art. Inducible promoters may be fused with the coding region of the DNA to provide an experimental means to regulate expression of the trans- gene. Alternatively or in addition, tissue specific regulatory elements may be fused with the coding region to permit tissue-specific expression of the trans-gene.
- microinjection needle which may be made from capillary tubing using a pipet puller
- the egg to be injected is put in a depression slide.
- the needle is inserted into the pronucleus of the egg, and the DNA solution is injected.
- the injected egg is then transferred into the oviduct of a pseudopregnant mouse (a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant), where it proceeds to the uterus, implants, and develops to term.
- pseudopregnant mouse a mouse stimulated by the appropriate hormones to maintain pregnancy but which is not actually pregnant
- This invention also provides a method for determining whether a ligand can specifically bind to a Y5 receptor which comprises contacting a cell transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction prepared from a cell extract of such cells, with the ligand under conditions permitting binding of ligands to such receptor, detecting the presence of any such ligand specifically bound to the Y5 receptor, and thereby determining whether the ligand specifically binds to the Y5 receptor.
- This invention provides a method for determining whether a ligand can specifically bind to a Y5 receptor which comprises contacting a cell transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the ligand under conditions permitting binding of ligands to such receptor, detecting the presence of any such ligand specifically bound to the Y5 receptor, and thereby determining whether the ligand specifically binds to the Y5 receptor, wherein the Y5 receptor has substantially the same amino acid sequence shown in Figure 6.
- This invention provides a method for determining whether a ligand can specifically bind to a Y5 receptor which comprises contacting a cell transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction of a cell extract of such cells, with the ligand under conditions permitting binding of ligands to such receptor, detecting the presence of any such ligand specifically bound to the Y5 receptor, and thereby determining whether the ligand specifically binds to the
- Y5 receptor wherein the Y5 receptor is characterized by an amino acid sequence in the transmembrane region having 60% homology or higher to the amino acid sequence in the transmembrane region of the Y5 receptor shown in Figure
- the Y5 receptor is a human Y5 receptor. In another embodiment of the above methods, the Y5 receptor is a rat Y5 receptor. In still another embodiment of the above methods, the Y5 receptor is a canine Y5 receptor.
- This invention provides a method for determining whether a ligand is a Y5 receptor agonist which comprises contacting a cell transfected with and expressing a Y5 receptor, or a membrane frction from a cell extract of such cells, with the ligand under conditions permitting activation of a functional Y5 receptor response, detecting a functional increase in Y5 receptor activity, and thereby determining whether the ligand is a Y5 receptor agonist.
- This invention further provides a method for determining whether a ligand is a Y5 receptor agonist which comprises contacting a cell transfected with and expressing a Y5 receptor, or a membrane fraction prepared from a cell extract of such cells, with the ligand under conditions permitting activation of the Y5 receptor, detecting an increase in Y5 receptor activity, and thereby determining whether the ligand is a Y5 receptor agonist.
- the Y5 receptor is a human Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5 receptor. In a further embodiment, the Y5 receptor is a canine Y5 receptor.
- This invention also provides a method for determining whether a ligand is a Y5 receptor antagonist which comprises contacting a cell transfected with and expressing nucleic acid encoding a Y5 receptor, or a membrane fraction from a cell extract of such cells, with the ligand in the presence of a known Y5 receptor agonist, such as PYY or NPY, under conditions permitting the activation of a functional Y5 receptor response, detecting a decrease in Y5 receptor activity, and thereby determining whether the ligand is a Y5 receptor antagonist.
- a known Y5 receptor agonist such as PYY or NPY
- This invention further provides a method for determining whether a ligand is a Y5 receptor antagonist which comprises contacting a cell transfected with and expressing DNA encoding a Y5 receptor, or a membrane fraction from a cell extract of such cells, with the ligand in the presence of a known Y5 receptor agonist, such as PYY or NPY, under conditions permitting the activation of the Y5 receptor, detecting a decrease in Y5 receptor activity, and thereby determining whether the ligand is a Y5 receptor antagonist.
- a known Y5 receptor agonist such as PYY or NPY
- the Y5 receptor is a human Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5 receptor. In a further embodiment, the Y5 receptor is a canine Y5 receptor.
- the cell is a Xenopus cell such as an oocyte or melanophore cell. In another embodiment of the methods described herein, the cell is a neuronal cell such as the glial cell line C6. In yet another embodiment of the methods described herein, the cell is non-neuronal in origin.
- the non-neuronal cell is a COS-7 cell, CHO cell, 293 human embryonic kidney cell, NlH-3T3 cell or LM(tk-) cell.
- the cell may be an insect cell such as a Sf-9 cell or Sf-21 cell.
- the ligand is not previously known.
- This invention additionally provides a Y5 receptor agonist detected by the above-described method.
- This invention also provides a Y5 receptor antagonist detected by the above-described method.
- This invention provides a method of screening a plurality of chemical compounds not known to bind to a Y5 receptor to identify a compound which specifically binds to the Y5 receptor which comprises (a) contacting a cell transfected with and expressing DNA encoding the Y5 receptor, or a membrane fraction from a cell extract of such cells, with a compound known to bind specifically to the Y5 receptor; (b) contacting the preparation of step (a) with the plurality of compounds not known to bind specifically to the Y5 receptor, under conditions permitting binding of compounds known to bind to the Y5 receptor; (c) determining whether the binding of the compound known to bind to the Y5 receptor is reduced in the presence of the compounds, relative to the binding of the compound in the absence of the plurality of compounds; and if so (d) separately determining the binding to the Y5 receptor of each compound included in the plurality of compounds, so as to thereby identify the compound which specifically binds to the Y5 receptor.
- Such competitive binding assays provide an efficient means to assess the receptor binding of chemical compounds either singly or in mixtures such as may be present in extracts of natural products or generated using combinatorial chemical synthetic methods for the production of peptidyl and non-peptidyl compounds.
- This invention provides a method of screening a plurality of chemical compounds not known to activate a Y5 receptor to identify a compound which activates the Y5 receptor which comprises (a) contacting a cell transfected with and expressing the Y5 receptor, or with a membrane fraction from a cell extract of such cells, with the plurality of compounds not known to bind specifically to the Y5 receptor, under conditions permitting activation of the Y5 receptor; (b) determining whether the activity of the Y5 receptor is increased in the presence of the compounds; and if so (c) separately determining whether the activation of the Y5 receptor is increased by each compound included in the plurality of compounds, so as to thereby identify the compound which activates the Y5 receptor.
- This invention further provides a method of screening a plurality of chemical compounds not known to inhibit the activation of a Y5 receptor to identify a compound which inhibits the activation of the Y5 receptor, which comprises (a) contacting a cell transfected with and expressing the Y5 receptor, or a membrane fraction from a cell exttact of such cells, with the plurality of compounds in the presence of a known Y5 receptor agonist, under conditions permitting activation of the Y5 receptor; (b) determining whether the activation of the Y5 receptor is reduced in the presence of the plurality of compounds, relative to the activation of the Y5 receptor in the absence of the plurality of compounds; and if so (c) separately determining the inhibition of activation of the Y5 receptor for each compound included in the plurality of compounds, so as to thereby identify the compound which inhibits the activation of the Y5 receptor.
- the Y5 receptor is a human Y5 receptor. In another embodiment, the Y5 receptor is a rat Y5 receptor. In a further embodiment, the Y5 receptor is a canine Y5 receptor. In an embodiment of the methods described herein, the cell is a Xenopus cell such as an oocyte or melanophore cell. In another embodiment, the cell is a mammalian cell. In a further embodiment, the mammalian cell is non-neuronal in origin. The cell may be a COS-7 cell, CHO cell, a 293 human embryonic kidney cell, a LM(tk-) cell, or an N1H- 3T3 cell.
- the cell is an insect cell such as a Sf-9 cell, Sf-21 cell, or HighFive cell.
- this invention provides a method of screening drugs to identify drugs which specifically bind to a Y5 receptor on the surface of a cell which comprises contacting a cell transfected with and expressing DNA encoding a Y5 receptor, or a membrane fraction from a cell extract of such cells, with a plurality of drugs under conditions permitting binding of drugs to the Y5 receptor, determining those drugs which specifically bind to the transfected cell, and thereby identifying drugs which specifically bind to the Y5 receptor.
- This invention provides a method of screening drugs to identify drugs which act as agonists of a Y5 receptor which comprises contacting a cell transfected with and expressing DNA encoding a Y5 receptor with a plurality of drugs under conditions permitting the activation of a functional Y5 receptor response, determining those drugs which activate such receptor in the .cell, and thereby identify drugs which act as Y5 receptor agonists.
- This invention provides a method of screening drugs to identify drugs which act as Y5 receptor antagonists which comprises contacting cells transfected with and expressing DNA encoding a Y5 receptor, or a membrane fraction from a cell extract of such cells, with a plurality of drugs in the presence of a known Y5 receptor agonist, such as PYY or NPY, under conditions permitting the activation of a functional Y5 receptor response, determining those drugs which inhibit the activation of the receptor in the mammalian cell, and thereby identifying drugs which act as Y5 receptor antagonists.
- the cell is a mammalian cell.
- the cell is nonneuronal in origin.
- This invention also provides a process for identifying a chemical compound which specifically binds to a Y5 receptor, which comprises contacting nonneuronal cells expressing on their cell surface the Y5 receptor, or a membrane fraction from a cell extract of such cells, with the chemical compound under conditions suitable for binding, and detecting specific binding of the chemical compound to the Y5 receptor.
- This invention further provides a process involving competitive binding for identifying a chemical compound which specifically binds to a Y5 receptor which comprises separately contacting nonneuronal cells expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with both the chemical compound and a second chemical compound known to bind to the receptor, and with only the second chemical compound, under conditions suitable for binding of both compounds, and detecting specific binding of the chemical compound to the Y5 receptor, a decrease in the binding of the second chemical compound to the Y5 receptor in the presence of the chemical compound indicating that the chemical compound binds to the Y5 receptor.
- This invention additionally provides a process for determining whether a chemcial compound specifically binds to and activates a Y5 receptor, which comprises contacting nonneuronal cells producing o second messenger response and expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with the chemical compound under conditions suitable for activation of the Y5 receptor, and measuring the second messenger response in the presence and in the absence of the chemical compound, a change in second messenger response in the presence of the chemical compound indicating that the chemical compound activates the Y5 receptor.
- This invention also provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a Y5 receptor, which comprises separately contacting nonneuronal cells producing a second messenger response and expressing on their cell surface a Y5 receptor, or a membrane fraction from a cell extract of such cells, with both the chemical compound and a second chemical compound known to activate the Y5 receptor, and with only the second chemical compound, under conditions suitable for activation of the Y5 receptor, and measuring the second messenger response in the presence of only the second chemical compound and in the presence of both the chemical compound and the second chemical compound, a smaller change in second messenger response in the presence of both the chemical compound and the second chemical compound indicating that the chemical compound inhibits activation of the Y5 receptor.
- the second messenger comprises adenylate cyclase activity and the change in second messenger response is a decrease in adenylate cyclase activity.
- the second messenger response comprises adenylate cyclase activity and the change in second messenger response is a smaller decrease in the level of adenylate cyclase activity in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- the second messenger comprises intracellular calcium levels and the change in second messenger response is an increase in intracellular calcium levels.
- the second messenger comprises intracellular calcium levels and the change in second messenger response is a smaller increase in the level of intracellular calcium in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- the cell is a mammalian cell.
- the cell is a COS-7 cell, a 293 human embryonic kidney cell, an LM(tk-) cell or an NlH-3T3 cell. It is further to be understood that any of the cells described herein, or any other appropriate host cell, may be used to express the Y5 receptors of the subject invention in any of the above-described embodiments.
- the Y5 receptor is a human Y5 receptor.
- the Y5 receptor is a rat or a canine Y5 receptor.
- binding and functional assays described herein may be performed using any cells which express the Y5 receptors of the subject invention, including, but not limited to, cells transfected with exogenous nucleic acid encoding Y5 receptors, as well as cultured cells or cell lines cultured under conditions which lead to expression of Y5 receptors detectable by either binding or functional assays.
- This invention also provides for any of the above methods for determining whether a compound activates or inhibits activation of any of the Y5 receptors described herein, wherein the activation is determined not by means of a second messenger response, but by effects of receptor activation which may occur prior to or independent of a second messenger response.
- measurement of the second messenger response is replaced with measurement of a change in the binding of GTP ⁇ S (a non- hydrolyzable analog of GTP) to cells transfected with and expressing a Y5 receptor or to a membrane fraction from such cells.
- the cells are nonneuronal cells.
- an increase in GTP ⁇ S binding to the cells or the membrane fraction in the presence of a compound indicates that the compound activates the Y5 receptor.
- a smaller increase in GTP ⁇ S binding to the cells or membrane fraction in the presence of both a compound known to activate the receptor and a test compound, relative to the increase in GTP ⁇ S binding in the presence of only the compound known to activate the receptor indicates that the test compound inhibits activation of the Y5 receptor.
- activation or inhibition of activation of any of the Y5 receptors disclosed herein may be measured by other means not requiring a second messneger, such as activation of MAP kinase, or activation of a reporter gene system, or by activation of immediate early genes, which are well known in the art.
- This invention provides a process for determining whether a chemical compound specifically binds to and activates a Y5 receptor, which comprises contacting nonneuronal cells expressing a Y5 receptor, or a membrane fraction from a cell extract of such cells, with the chemical compound under conditions suitble for activation of the Y5 receptor, and measuring the binding of GTP ⁇ S to the cells or membrane fraction, in the presence and in the absence of the chemical compound, a change in the binding of GTP ⁇ S in the presence of the chemical compound indicating that the chemical compound activates the Y5 receptor.
- This invention further provides a process for determining whether a chemical compound specifically binds to and inhibits activation of a Y5 receptor, which comprises separately contacting nonneuronal cells expressing a Y5 receptor, with both the chemical compound and a second chemical compound known to activate the Y5 receptor, and with only the second chemical compound, under conditions suitable for activation of the Y5 receptor, and measuring binding of GTP ⁇ S to the cell or membrane fraction in the presence of only the second chemical compound and in the presence of both the second chemical compound and the chemical compound, a smaller change in GTP ⁇ S binding in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound indicating that the chemical compound inhibits activation of a Y5 receptor.
- the change in binding is an increase in GTP ⁇ S binding.
- the change in binding is a smaller increase in GTP ⁇ S binding in the presence of both the chemical compound and the second chemical compound than in the presence of only the second chemical compound.
- the cells are not intact.
- the expression level of endogenous receptors can be increased several-fold by treatment with compounds such as Il-l ⁇ (Menke, et al., 1994), NGF (Dimaggio, et al., 1994) or glucocorticoids (Larsen, et al., 1994).
- compounds such as Il-l ⁇ (Menke, et al., 1994), NGF (Dimaggio, et al., 1994) or glucocorticoids (Larsen, et al., 1994).
- Such treatment may allow screening of compounds at Y5 receptors in cell lines expressing previously undetectable levels of endogenous Y5 receptors, without transfecting such cell lines with the Y5 receptor.
- One may also create recombinant cell lines, whereby the normal promoter may be replaced with promoter element (s) that allow increased expression of the Y5 gene, thereby allowing one to screen compounds using the recombinant cell line.
- Such cells and cell lines may be used with any
- This invention provides a pharmaceutical composition comprising a drug identified by the above-described methods and a pharmaceutically acceptable carrier.
- This invention provides a method of detecting expression of Y5 receptor by detecting the presence of mRNA coding for the Y5 receptor which comprises obtaining total mRNA from the cell and contacting the mRNA so obtained with the above-described nucleic acid probe under hybridizing conditions, detecting the presence of mRNA hybridized to the probe, and thereby detecting the expression of the Y5 receptor by the cell.
- This invention provides a method of treating obesity and other disorders associated with excess eating (e.g., bulimia) in which a Y5 receptor antagonist is administered in combination with existing therapies.
- a Y5 receptor antagonist is administered in combination with existing therapies.
- An example os such a drug is dexfenfluramine, a serotonin uptake inhibitor (McTavish, D. and R.C. Heel, Drugs 12(5) :713-733 (1992)).
- Administration of dexfenfluramine results in significant weight loss after about one month of therapy, with maximal weight loss occurring in the first six months of therapy. It is noteworthy that after discontinuation of drug therapy an increas in body weight is observed after about two months.
- This invention provides a method of decreasing feeding behavior in a subject which comprises administering to the subject a compound which is a Y5 receptor antagonist and a compound which is monoamine neurotransmitter uptake inhibitor, wherein the amount of the Y5 antagonist and the monoamine neurotransmitter uptake inhibitor are effective to decrease the feeding behavior of the subject.
- This invention also provides the use of a compound which is a Y5 receptor antagonist and a compound which is a monoamine neurotransmitter uptake inhibitor for the preparation of a pharmaceutical composition for decreasing feeding behavior in a subject, wherein the amount of the Y5 receptor antagonist and the amount of the monoamine neurotransmitter uptake inhibitor is effective to decrease feeding behavior in the subject.
- the Y5 receptor antagonist and the monoamine neurotransmitter uptake inhibitor aer administered in combination.
- the Y5 receptor antagonist and the monoamine neurotransmitter uptake inhibitor are administered once.
- the Y5 receptor antagonist and the monoamine neurotransmitter uptake inhibitor are administered separately.
- the Y5 receptor antagonist and the monoamine neurotransmitter uptake inhibitor are administered once.
- the Y5 receptor antagonist is administered for about two weeks to about six months.
- the monoamine neurotransmitter uptake inhibitor is administered for about one month to about six months.
- the Y5 receptor antagonist and the monoamine neurotransmitter uptake inhibitor are administered repeatedly.
- the Y5 receptor antagonist is administered for about two weeks to about six months.
- the monoamine neurotransmitter uptake inhibitor is administered for about one month to about six months.
- the neurotransmitter uptake inhibitor is administered for about one month to about three months.
- the monoamine neurotransmitter uptake inhibitor may be fenfluramine, dexfenfluramine, or sibutramine.
- the compound is administered in a pharmaceutical composition comprising a sustained release formula.
- This invention provides a method of decreasing feeding behavior of a subject which comprises administering to the subject a compound which is a galanin receptor antagonist and a compound which is a Y5 receptor antagonist, wherein the amount of the antagonists is effective to decrease feeding behavior of the subject.
- the galanin receptor antagonist and the Y5 receptor antagonist are administered in combination.
- the galanin receptor antagonist and the Y5 receptor antagonist are administered once.
- the galanin receptor antagonist and the Y5 receptor antagonist are administered separately.
- the galanin receptor antagonist and the Y5 receptor antagonist are administered once.
- the galanin receptor antagonist is administered for about 1 week to about 2 weeks.
- the Y5 receptor antagonist is administered for about 1 week to about 2 weeks.
- the galanin receptor antagonist and the Y5 receptor antagonist are administered repeatedly.
- the galanin receptor antagonist is administered for about 1 week to about 2 weeks.
- the galanin receptor is a GALR2 receptor or a GALR3 receptor.
- the compound is administered in a pharmaceutical composition comprising a sustained release formulation.
- This invention provides a method of treating an abnormality in a subject, wherein the abnormality is alleviated by the inhibition of a Y5 receptor which comprises administering to a subject an amount of the above-described pharmaceutical composition effective to decrease the activity of the Y5 receptor in the subject and thereby treat the abnormality.
- This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by the activation of a Y5 receptor which comprises administering to a subject an amount of the above-described pharmaceutical composition effective to incresase the activation of the Y5 receptor in the subject and thereby treate the abnormality.
- This invention provides a method of treating an abnormality in a subject, wherein the abnormality is alleviated by the decreasing the activity of a Y5 receptor which comprises administering to a subject an amount of the above-described pharmaceutical composition effective to decrease the activity of the Y5 receptor and thereby treat the abnormality.
- the abnormality is obesity.
- the abnormality is bulimia.
- This invention provides a method of treating an abnormality in a subject wherein the abnormality is alleviated by the activation of a Y5 receptor which comprises administering to a subject an effective amount of a Y5 receptor agonist.
- the abnormal condition is anorexia.
- the abnormal condition is a sexual/reproductive disorder.
- the abnormal condition is depression.
- the abnormal condition is anxiety.
- the abnormal condition is gastric ulcer. In a further embodiment, the abnormal condition is memory loss. In a further embodiment, the abnormal condition is migraine. In a further embodiment, the abnormal condition is pain. In a further embodiment, the abnormal condition is epileptic seizure. In a further embodiment, the abnormal condition is hypertension. In a further embodiment, the abnormal condition is cerebral hemorrhage. In a further embodiment, the abnormal condition is shock. In a further embodiment, the abnormal condition is congestive heart failure. In a further embodiment, the abnormal condition is sleep disturbance. In a further embodiment, the abnormal condition is nasal congestion. In a further embodiment, the abnormal condition is diarrhea.
- This invention further provides a method of treating obesity in a subject which comprises administering to the subject an effective amount of a Y5 receptor antagonist.
- This invention also provides a method of treating anorexia in a subject which comprises administering to the subject an effective amount of a Y5 receptor agonist.
- this invention provides a method of treating bulimia nervosa in a subject which comprises administering to the subject an effective amount of a Y5 receptor antagonist.
- This invention provides a method of inducing a subject to eat which comprises administering to the subject an effective amount of a Y5 receptor agonist.
- the subject is a vertebrate.
- the subject is a human.
- the subject is a rat.
- the subject is a canine subject.
- This invention also provides a method of increasing the consumption of a food product by a subject which comprises administering to the subject a composition of the food product and an amount of a Y5 receptor agonist.
- the subject is a vertebrate.
- the subject is a human, a rat or a canine subject.
- This invention also provides a method of treating abnormalities which are alleviated by reduction of activity of a human Y5 receptor which comprises administering to a subject an amount of the above- described pharmaceutical composition effective to reduce the activity of human Y5 receptor and thereby alleviate abnormalities resulting from overactivity of a human Y5 receptor.
- This invention further provides a method of treating an abnormal condition related to an excess of Y5 receptor activity which comprises administering to a subject an amount of the pharmaceutical composition effective to block binding of a ligand to the Y5 receptor and thereby alleviate the abnormal condition.
- This invention additionally provides a method of detecting the presence of a Y5 receptor on the surface of a cell which comprises contacting the cell with the antibody capable of binding to the Y5 receptor under conditions permitting binding of the antibody to the receptor, detecting the presence of the antibody bound to the cell, and thereby detecting the presence of a Y5 receptor on the surface of the cell.
- This invention also provides a method of determining the physiological effects of varying levels of activity of a Y5 receptor which comprises producing a transgenic nonhuman mammal whose levels of Y5 receptor activity are varied by use of an inducible promoter which regulates Y5 receptor expression.
- This invention further provides a method of determining the physiological effects of varying levels of activity of a Y5 receptors which comprises producing a panel of transgenic nonhuman mammals each expressing a different amount of Y5 receptor.
- This invention provides a method for identifying a substance capable of alleviating the abnormalities resulting from overactivity of a Y5 receptor comprising administering a substance to the above-described transgenic nonhuman mammals, and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of overactivity of a Y5 receptor.
- This invention also provides a method for treating abnormalities resulting from overactivity of a Y5 receptor which comprises administering to a subject an amount of the above-described pharmaceutical composition effective reduce the activation of the Y5 receptor and thereby alleviate the abnormalities resulting from overactivity of a Y5 receptor.
- This invention further provides a method for identifying a substance capable of alleviating the abnormalities resulting from underactivity of a Y5 receptor comprising administering the substance to the above-described transgenic nonhuman mammals and determining whether the substance alleviates the physical and behavioral abnormalities displayed by the transgenic nonhuman mammal as a result of underactivity of a Y5 receptor.
- This invention additionally provides a method for treating the abnormalities resulting from underactivity of a Y5 receptor which comprises administering to a subject an amount of the above-described pharmaceutical composition effective to increase the activation of the Y5 receptor and thereby alleviate the abnormalities resulting from underactivity of a Y5 receptor.
- This invention provides a method for diagnosing a predisposition to a disorder associated with the activity of a specific allelic form of a Y5 receptor which comprises: a. obtaining DNA from the subject to be tested; digesting the DNA with restriction enzymes; c. separating the resulting DNA fragments; d. contacting the fragments with a detectably labeled nucleic acid probe capable of specifically hybridizing with a sequence uniquely present within the sequence of a nucleic acid encoding the allelic form of the Y5 receptor; and e. detecting the presence of labeled probe hybridized to the DNA fragments from the subject being tested, the presence of such hybridized probe indicating that the subject is predisposed to the disorder.
- This invention also provides a method of preparing an isolated Y5 receptor which comprises: a. inducing cells to express the Y5 receptor; b. recovering the receptor from the resulting cells; and c. purifying the receptor so recovered.
- This invention further provides a method of preparing the isolated Y5 receptor which comprises: a. inserting nucleic acid encoding Y5 receptor in a suitable vector adapted for expression in a bacterial, yeast, insect, or mammalian cell operatively linked to the nucleic acid encoding the Y5 receptor as to permit expression thereof; b. inserting the resulting vector in a suitable host cell so as to obtain a cell which produces the Y5 receptor; c. recovering the receptor produced by the resulting cell; and d. purifying the receptor so recovered.
- This invention provides a method for detecting in a subject the presence of a restriction fragment length polymorphism associated with a genomic locus which encompasses both a Y1 and a Y5 receptor gene which comprises: a) obtaining a sample of DNA from the subject; b) digesting the DNA with a restriction enzyme; c) separating the resulting DNA fragments; d) contacting the DNA fragments with a detectably labeled nucleic acid probe which specifically hybridizes with a sequence uniquely present within the sequence associated with the polymorphism; and e) detecting whether the probe hybridizes to the DNA fragments, the presence of the labeled probe hybridized to the DNA fragment indicating the presence of the restriction fragment length polymorphism.
- the restriction enzyme is PstI .
- the subject is a human.
- the PstI polymorphism is associated with susceptibility to modification of feeding behavior using a Y5-selective compound.
- the feeding behavior is anorexia or bulimia, or the feeding behavior is associated with obesity.
- the subject is a human.
- the subject is a non-human animal.
- the subject is a mammal.
- the subject is a bovine, equine, canine or feline.
- This invention provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the binding of the compound to the human Y5 receptor is characterized by a K i less than 100 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration, and wherein the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor.
- the binding of the compound to each of the human Y1, human Y2, and human Y4 receptors is characterized by a K i greater than 500 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration. In another embodiment, the binding of the compound to each of the human Y1, human Y2, and human Y4 receptors is characterized by a K i greater than 1000 nanomolar.
- This invention also provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the binding of the compound to the human Y5 receptor is characterized by a K i less than 5 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the compound to each of the human Y1, human Y2, and human Y4 receptors is characterized by a K i greater than 5 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor.
- the binding of the compound to each of the human Y1, human Y2 and human Y4 receptors is characterized by a K i greater than 50 nanomolar when measured in the presence of 125 I-PYY at a predetermined concentration.
- the binding of the compound to each of the human Y1, human Y2 and human Y4 receptors is characterized by a K i greater than 100 nanomolar.
- This invention further provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and greater than 26-fold higher than the affinity with which the compound binds to the human Y1 receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and greater than 22-fold higher than the affinity with which the compound binds to the human Y2 receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and greater than 34-fold higher than the affinity with which the compound binds to the human Y4 receptor.
- the compound binds to the human Y5 receptor with an affinity a) greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor; b) greater than 22-fold higher than the affinity with which the compound binds to the human Y2 receptor; and c) greater than 34-fold higher than the affinity with which the compound binds to the human Y4 receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and with an affinity a) greater than 26- fold higher than the affinity with which the compound binds to the human Y1 receptor; b) greater than 22-fold higher than the affinity with which the compound binds to the human Y2 receptor; and c) and greater than 34-fold higher than the affinity with which the compound binds to the human Y4 receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and greater than 100-fold higher than the affinity with which the compound binds to the human Y1 receptor. In a further embodiment of the above described methods, the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and greater than 165-fold higher than the affinity with which the compound binds to the human Y2 receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and greater than 143-fold higher than the affinity with which the compound binds to the human Y4 receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor and a) greater than 143-fold higher than the affinity with which the compound binds to the human Y4 receptor; and b) greater than 165-fold higher than the affinity with which the compound binds to the human Y2 receptor.
- the compound binds to the human Y5 receptor with an affinity greater than ten-fold higher than the affinity with which the compound binds to any other human Y-type receptor, and a) greater than 143-fold higher than the affinity with which the compound binds to the human Y4 receptor; b) greater than 165-fold higher than the affinity with which the compound binds to the human Y2 receptor; and c) greater than 100-fold higher than the affinity with which the compound binds to the human Y1 receptor.
- This invention additionally provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the compound binds to the human Y5 receptor with an affinity greater than 500-fold higher than the affinity with which the compound binds to each of the human Y1, human Y2, and human Y4 receptors.
- This invention also provides a method of treating a subject's feeding disorder which comprises administering to the subject a non-peptidyl compound which is a Y5 receptor antagonist in an amount effective to inhibit the activity of the subject's Y5 receptor, wherein the compound binds to the human Y5 receptor with an affinity greater than 1400-fold higher than the affinity with which the compound binds to each of the human Y1, human Y2, and human Y4 receptors.
- the feeding disorder is obesity or bulimia.
- the subject is a vertebrate, a mammal, a human or a canine.
- RNA was prepared by a modification of the guanidine thiocyanate method (Kingston, 1987), from 5 grams of rat hypothalamus (Rockland, Gilbertsville, PA). Poly A + RNA was purified with a FastTrack kit (Invitrogen Corp., San Diego, CA). Double stranded (d ⁇ ) cDNA was synthesized from 7 ⁇ g of poly A + RNA according to Gubler and Hoffman (Gubler and Hoffman, 1983), except that ligase was omitted in the second strand cDNA synthesis.
- the resulting ds-cDNA was ligated to BstXI/EcoRI adaptors (Invitrogen Corp.), the excess of adaptors was removed by chromatography on Sephacryl 500 HR (Pharmacia ® -LKB) and the ds-cDNA size selected on a Gen-Pak Fax HPLC column (Millipore Corp., Milford, MA). High molecular weight fractions were ligated in pEXJ.BS (A cDNA cloning expression vector derived from pcEXV-3 ; Okayama and Berg, 1983; Miller and Germain, 1986) cut by BstXI as described by Aruffo and Seed (Aruffo and Seed, 1987).
- the ligated DNA was electroporated in E.coli MC 1061 F + (Gene Pulser, Biorad). A total of 3.4 x 10 6 independent clones with an insert mean size of 2.7 kb could be generated.
- the library was plated on Petri dishes (Ampicillin selection) in pools of 6.9 to 8.2 x 10 3 independent clones. After 18 hours amplification, the bacteria from each pool were scraped, resuspended in 4 mL of LB media and 1.5 mL processed for plasmid purification with a QIAprep-8 plasmid kit (Qiagen Inc, Chatsworth, CA). 1 ml aliquots of each bacterial pool were stored at -85°C in 20% glycerol.
- COS-7 cells DNA from pools of ⁇ 7500 independent clones was transfected into COS-7 cells by a modification of the DEAE-dextran procedure (Warden and Thorne, 1968).
- COS-7 cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 2mM L-glutamine (DMEM-C) at 37 °C in 5% CO 2 .
- DMEM Dulbecco's modified Eagle medium
- the cells were seeded one day before transfection at a density of 30,000 cells/cm 2 on Lab-Tek chamber slides (1 chamber, Permanox slide from Nunc Inc., Naperville, IL).
- rat oligonucleotide primers in TM 3 sense primer; position 484-509 in fig. IA
- TM 6 antisense primer; position 1219-1243 in fig. 3A
- a human hippocampal cDNA library using the polymerase chain reaction.
- 1 ⁇ l (4 x 10 6 bacteria) of each of 450 amplified pools containing each ⁇ 5000 independent clones and representing a total of 2.2 x 10 6 was subjected directly to 40 cycles of PCR and the resulting products analyzed by agarose gel electrophoresis.
- One of three positive pools was analyzed further and by sib selection a single cDNA clone was isolated and characterized. This cDNA turned out to be full length and in the correct orientation for expression.
- DS-DNA was sequenced with a sequenase kit
- the primers CH156-CH153 were used to amplify 10 ng of poly (A+) RNA from rat brain that was reverse transcribed using the SSII reverse transcriptase (GibcoBRL, Gaithersburg, MD). PCR was performed on single-stranded cDNA with Taq Polymerase (Perkin Elmer-Roche Molecular Systems, Branchburg, NJ) under the following conditions: 94°C for 1 min, 60°C for 1 min and 72°C for 1 min for 40 cycles.
- the resulting 798 bp PCR DNA fragment was subcloned in pCR Script (Stratagene, La Jolla, CA) and sequenced using a sequenase kit (USB, Cleveland, OH) and is designated Y5-bd-5.
- CH 245 (nested primer): 5'-TTCTTGAGTGGTTCTCTTGAGGAGG-3' (Seq. I.D. No. 12).
- the 3' and 5' RACE reactions were carried out on canine thalamic cDNA according to the kit specifications, with the primers described above.
- the resulting PCR DNA products (smear of 0.7 to 10 kb) were purified from an agarose gel and reamplified using the nested primers described above.
- the resulting discrete DNA bands were again purified from an agarose gel and subcloned in pCR Script (Stratagene, La Jolla, CA).
- the nucleotide sequence corresponding to the 3' end of the cDNA was determined and the plasmid designated Y5-bd- 8. However, attempts to determine the 5' sequence of the beagle Y5 receptor by 5' RACE were unsuccessful.
- a canine brain cDNA library (in the pEXJ vector) was screened by PCR using primers BB33 (TM-3) and BB34 (3-4 loop).
- Vector-anchored PCR using primers BB34 and KS938 (pEXJ + strand) or KS939 (pEXJ - strand) was then used to amplify the 5' end from two positive pools.
- the resulting PCR products (0.6 and 0.57 kb) were purified from an agarose gel and subcloned into the pCR Script vector (Stratagene, La Jolla, CA).
- nucleotide sequence of the longer of these products was determined using a sequenase kit (USB, Cleveland, OH) and designated dogY5-16.
- dogY5-16 By comparison to the human Y5 receptor, dogY5-16 lacked the first 18 nucleotides of the Y5 coding sequence.
- a nitrocellulose membrane (Schleicher and Schuell, Keene, NH) containing 20 ⁇ g of Hindlll-cut canine genomic DNA (Clontech, Palo Alto, CA) was hybridized with a 32 P-labeled oligonucleotide probe (BB53) corresponding to nucleotides 3-35 of dogY5-16.
- BB53 32 P-labeled oligonucleotide probe
- a 4.2 kb hybridizing band was isolated from a replicate agarose gel and subcloned into the pUC18 vector.
- Vector anchored PCR was performed on one-tenth of the ligation reaction using BB34 (3-4 loop) and BB77 (pUCl8 + strand) or BB78 (pUC18 - strand).
- the resulting PCR products (1.35, 0.87, 0.75 and 0.7 kb) were then re-amplified using BB77 and a nested primer BB70 (nucleotides 94-111 from dogY5-16).
- the resulting PCR products (0.4, 0.7 and 0.95 kb) were purified from an agarose gel and subcloned in pCR Script (Stratagene, La Jolla, CA). A portion of the 0.95 kb fragment, designated dogY5-2-29, was sequenced using a Sequenase kit (USB, Cleveland, OH).
- the primers BB80 (5' untranslated sequence ⁇ UT ⁇ from dogY5-2-29) and BB54 (carboxy tail and 3' UT from Y5-bd-8) were used to amplify 0.36 ⁇ g of beagle genomic DNA.
- PCR was performed using Expand High Fidelity polymerase (Boehringer Mannheim Corporation, Indianapolis, 1N) under the following conditions: 94°C for 1 min, 63°C for 2 min and 68 °C for 3 min for 38 cycles.
- the resulting 1.4 kb PCR band was purified from an agarose gel and subcloned into pEXJ.
- Three clones, designated BO10, B011 and B012 were sequenced using a sequenase kit (USB, Clevland, OH).
- the pEXJ derived plasmid comprising clone BO11 was designated CY5-BO11 and was deposited with the ATCC on May 29, 1996, under ATCC Accession No. 97587.
- Human brain multiple tissue northern blots (MTN blots II and III, Clontech, Palo Alto, CA) carrying mRNA purified from various human brain areas was hybridized at high stringency according to the manufacturer specifications.
- the probe was a 0.8 kb DNA PCR fragment corresponding to the TM III - carboxy end of the 5-6 loop in the coding region of the human Y5 receptor subtype.
- a rat multiple tissue northern blot (rat MTN blot, Clontech, Palo Alto, CA) carrying mRNA purified from various rat tissues was hybridized at high stringency according to the manufacturer specifications.
- the probe was a 0.8 kb DNA PCR fragment corresponding to the TM III - carboxy end of the 5-6 loop in the coding region of the rat Y5 receptor subtype.
- Southern blots (Geno-Blot, clontech, Palo Alto, CA) containing human or rat genomic DNA cut with five different enzymes (8 ⁇ g DNA per lane) was hybridized at high stringency according to the manufacturer specifications.
- the probe was a 0.8 kb DNA PCR fragment corresponding to the TM III - carboxy end of the 5-6 loop in the coding region of the human and rat Y5 receptor subtypes.
- a BamHI site directly 5' to the starting methionine of human Y5 was genetically engineered by replacing the beginning «100 base pairs of hY5 (i.e. from the starting methionine to an internal EcoRI site) with two overlapping synthetically-derived oligonucleotides ( ⁇ 100 bases each), containing a 5' BamHI site and a 3' EcoRI site. This permitted the isolation of an ⁇ 1.5 kb Bam Hi/Hind III fragment containing the coding region of hY5. This fragment was subcloned into pBlueBacIIlTM into the Bam Hi/Hind III sites found in the polylinker (construct called pBB/hY5).
- baculovirus 0.5 ⁇ g of viral DNA (BaculoGoldTM) and 3 ⁇ g of pBB/hY5 were co- transfected into 2 x 10 6 Spodoptera frugiperda insect Sf9 cells by calcium phosphate co-precipitation method, as outlined by Pharmingen (in "Baculovirus Expression Vector System: Procedures and Methods Manual”). The cells were incubated for 5 days at 27°C. The supernatant of the cotransfection plate was collected by centrifugation and the recombinant virus (hY5BB3) was plaque purified. The procedure to infect cells with virus, to prepare stocks of virus and to titer the virus stocks were as described in Pharmingen's manual. Cell Culture
- COS-7 cells were grown on 150 mm plates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37 °C, 5% C0 2 . Stock plates of COS-7 cells were trypsinized and split 1:6 every 3-4 days.
- D-MEM Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin
- Human embryonic kidney 293 cells were grown on 150 mm plates in D-MEM with supplements (minimal essential medium) with Hanks' salts and supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37°C, 5% CO 2 . Stock plates of 293 cells were trypsinized and split 1:6 every 3-4 days.
- Mouse fibroblast LM(tk-) cells were grown on 150 mm plates in D-MEM with supplements (Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mM glutamine, 100 units/mL penicillin/100 ⁇ g/mL streptomycin) at 37°C, 5% CO 2 . Stock plates of LM(tk-) cells were trypsinized and split 1:10 every 3-4 days.
- LM(tk-) cells stably transfected with the human Y5 receptor were routinely converted from an adherent monolayer to a viable suspension.
- Adherent cells were harvested with trypsin at the point of confluence, resuspended in a minimal volume of complete DMEM for a cell count, and further diluted to a concentration of 10 6 cells/ml in suspension media (10% bovine calf serum, 10% 10X Medium 199 (Gibco), 9 mM NaHCO 3 , 25 mM glucose, 2 mM L-glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin, and 0.05% methyl cellulose).
- the cell suspension was maintained in a shaking incubator at 37 °C, 5% CO 2 for 24 hours.
- Membranes harvested from cells grown in this manner may be stored as large, uniform batches in liquid nitrogen.
- cells may be returned to adherent cell culture in complete DMEM by distribution into 96-well microtiter plates coated with poly-D-lysine (0.01 mg/ml) followed by incubation at 37 °C, 5% CO 2 for 24 hours.
- Cells prepared in this manner yielded a robust and reliable NPY-dependent response in cAMP radio-immunoassays as further described hereinbelow.
- Mouse embryonic fibroblast NlH-3T3 cells were grown on 150 mm plates in Dulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovine calf serum, 4 mM glutamine, 100 units/ml penicillin/100 ⁇ g/ml streptomycin) at 37 °C, 5% CO 2 . Stock plates of NlH-3T3 cells were trypsinized and split 1:15 every 3-4 days.
- DMEM Dulbecco's Modified Eagle Medium
- Sf9 and Sf21 cells were grown in monolayers on 150 mm tissue culture dishes in TMN-FH media supplemented with 10% fetal calf serum, at 27 °C, no CO 2 .
- High Five insect cells were grown on 150 mm tissue culture dishes in Ex- Cell 400TM medium supplemented with L-Glutamine, also at 27°C, no CO 2 .
- Human Y1, human Y2, and rat Y5 receptors were co- transfected with a G-418 resistant gene into the human embryonic kidney 293 cell line by a calcium phosphate transfection method (Cullen, 1987). Stably transfected cells were selected with G-418.
- Human Y4 and human Y5 receptors were similarly transfected into mouse fibroblast LM(tk-) cells and N1H-3T3 cells.
- Canine Y5 receptors also may be similarly transfected into LM(tk-), N1H-3T3 cells or other appropriate host cells.
- Additional host cells appropriate for transfection of the Y-type receptors include, but are not limited to, Chinese hamster ovary cells (CHO), the glial cell line C6, or non-mammalian host cells such as Xenopus melanophore cells.
- CHO Chinese hamster ovary cells
- C6 the glial cell line C6
- non-mammalian host cells such as Xenopus melanophore cells.
- ⁇ 1 Human Adrenergic Receptors To determine the binding of compounds to human ⁇ 1 receptors, LM(tk-) cell lines stably transfected with the genes encoding the ⁇ 1a , ⁇ 1b , and ⁇ 1d receptors were used. The nomenclature describing the ⁇ , receptors was changed recently, such that the receptor formerly designated ⁇ 1a is now designated ⁇ ld , and the receptor formerly designated ⁇ 1c is now designated ⁇ 1 (ref). The cell lines expressing these receptors were deposited with the ATCC before the nomenclature change and reflect the subtype designations formerly assigned to these receptors.
- the cell line expressing the receptor described herein as the ⁇ 1a receptor was deposited with the ATCC on September 25, 1992, under ATCC Accession No. CRL 11140 with the designation L- ⁇ 1c .
- the cell line expressing receptor described herein as the ⁇ 1d receptor was deposited with the ATCC on September 25, 1992, under ATCC Accession No. CRL 11138 with the designation L- ⁇ 1A .
- the cell line expressing the ⁇ 1b receptor is designated L- ⁇ 1B , and was deposited on September 25, 1992, under ATCC Accession No. CRL 11139.
- LM(tk-) cell lines stably transfected with the genes encoding the ⁇ 2A , ⁇ 2B , and ⁇ 2C receptors were used.
- the cell line expressing the ⁇ 2A receptor is designated L- ⁇ 2A , and was deposited on November 6, 1992, under ATCC Accession No. CRL 11180.
- the cell line expressing the ⁇ 2B receptor is designated L- NGC- ⁇ 2B , and was deposited on October 25, 1989, under ATCC Accession No. CRL 10275.
- the cell line expressing the ⁇ 2c receptor is designated L- ⁇ 2C , and was deposited on November 6, 1992, under ATCC Accession No.
- CRL-11181 Cell lysates were prepared as described below (see Radioligand Binding to Membrane Suspensions), and suspended in 25mM glycylglycine buffer (pH 7.6 at room temperature). Equilibrium competition binding assay were performed using [ 3 H]rauwolscine (0.5nM), and nonspecific binding was determined by incubation with 10 ⁇ M phentolamine. The bound radioligand was separated by filtration through GF/B filters using a cell harvester.
- Human Histamine H, Receptor The coding sequence of the human histamine H, receptor, homologous to the bovine H 1 receptor, was obtained from a human hippocampal cDNA library, and was cloned into the eukaryotic expression vector pcEXV-3.
- the plasmid DNA for the H 1 receptor is designated pcEXV-H1, and was deposited on November 6, 1992, under ATCC Accession No. 75346. This construct was transfected into COS-7 cells by the DEAE-dextran method. Cells were harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM EDTA, pH 7.5.
- the cell lysates were centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant was centrifuged at 30,000 x g for 20 min. at 4°C.
- the pellet was suspended in 37.8 mM NaHPO A , 12.2 mM KH 2 PO 4 , pH 7.5.
- the binding of the histamine H 1 antagonist [ 3 H]mepyramine was done in a final volume of 0.25 mL and incubated at room temperature for 60 min. Nonspecific binding was determined in the presence of 10 ⁇ M mepyramine.
- the bound radioligand was separated by filtration through GF/B filters using a cell harvester.
- Human Histamine H 2 Receptor The coding sequence of the human H 2 receptor was obtained from a human placenta genomic library, and cloned into the cloning site of PCEXV-3 eukaryotic expression vector.
- the plasmid DNA for the H 2 receptor is designated pcEXV-H2, and was deposited on November 6, 1992 under ATCC Accession No. 75345. This construct was transfected into COS-7 cells by the DEAE-dextran method. Cells were harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM EDTA, pH 7.5.
- the cell lysates were centrifuged at 1000 rpm for 5 min at 4°C, and the supernatant was centrifuged at 30,000 x g for 20 min at 4 °C.
- the pellet was suspended in 37.8 mM NaHPO 4 , 12.2 mM K 2 PO 4 , pH 7.5.
- the binding of the histamine H 2 antagonist [ 3 H] tiotidine was done in a final volume of 0.25 ml and incubated at room temperature for 60 min. Nonspecific binding was determined in the presence of 10 ⁇ M histamine.
- the bound radioligand was separated by filtration through GF/B filters using a cell harvester.
- 5HT 1Da , 5HT 1D ⁇ , 5HT 1E , 5HT 1F Receptors LM(tk-) clonal cell lines stably transfected with the genes encoding each of these 5HT receptor subtypes were prepared as described above.
- the cell line for the 5HT 1D ⁇ receptor designated as Ltk-8-30-84, was deposited on April 17, 1990, and accorded ATCC Accession No. CRL 10421.
- the cell for the 5HT 1D ⁇ receptor, designated as Ltk-11 was deposited on April 17, 1990, and accorded ATCC Accession No. CRL 10422.
- the cell line for the 5HT 1E receptor designated 5 HT 1E -7, was deposited on November 6, 1991, and accorded ATCC Accession No. CRL 10913.
- the cell line for the 5HT 1F receptor designated L-5-HT 1F , was deposited on December 27, 1991, and accorded ATCC Accession No. ATCC 10957.
- Membrane preparations comprising these receptors were prepared as described below, and suspended in 50mM Tris- HCl buffer (pH 7.4 at 37°C) containing 10 mM MgCl 2 , 0.2 mM EDTA, 10 ⁇ M pargyline, and 0.1% ascorbate.
- the binding of compounds was determined in competition binding assays by incubation for 30 minutes at 37°C in the presence of 5nM [ 3 H] serotonin. Nonspecific binding was determined in the presence of 10 ⁇ M serotonin.
- the bound radioligand was separated by filtration through GF/B filters using a cell harvester.
- Human 5HT 2 Receptor The coding sequence of the human 5HT 2 receptor was obtained from a human brain cortex cDNA library, and cloned into the cloning site of pcEXV-3 eukaryotic expression vector. This construct was transfected into COS-7 cells by the DEAE-dextran method. Cells were harvested after 72 hours and lysed by sonication in 5mM Tris-HCl, 5mM EDTA, pH 7.5. This cell line was deposited with the ATCC on October 31, 1989, designated as L-NGC-5HT 2 , and was accorded ATCC Accession No. CRL 10287.
- the cell lysates were centrifuged at 1000 rpm for 5 minutes at 4°C, and the supernatant was centrifuged at 30,000 x g for 20 minutes at 4°C.
- the pellet was suspended in 50mM Tris-HCl buffer (pH 7.7 at room temperature) containing 10 mM MgSO 4 , 0.5mM EDTA, and 0.1% ascorbate.
- the potency of alpha-1 antagonists at 5HT 2 receptors was determined in equilibrium competition binding assays using [3H]ketanserin (1nM). Nonspecific binding was defined by the addition of 10 ⁇ M mianserin.
- the bound radioligand was separated by filtration trrough GF/B filters using a cell harvester.
- Human 5-HT 7 Receptor A LM(tk-) clonal cell line stably transfected with the gene encoding the 5HT 7 receptor subtype was prepared as described above. The cell line for the 5HT 7 receptor, designated as L-5HT 4B; was deposited on October 20, 1992, and accorded ATCC Accession No. CRL 11166.
- Human Dopamine D 3 Receptor The binding of compounds to the human D3 receptor was determined using membrane preparations from COS-7 cells transfected with the gene encoding the human D 3 receptor. The human dopamine D3 receptor was prepared using known methods. Sokoloff, P.
- Membranes were harvested from COS-7 cells 48 hours after transient transfection.
- Adherent cells were washed twice in ice-cold phosphate buffered saline (138 mM NaCl, 8.1 mM Na 2 HPO 4 , 2.5 mM KCl, 1.2 mM KH 2 PO 4 , 0.9 mM CaCl 2 , 0.5 mM MgCl 2 , pH 7.4) and lysed by sonication in ice-cold sonication buffer (20 mM Tris-HCl, 5 mM EDTA, pH 7.7). Large particles and debris were cleared by low speed centrifugation (200 x g, 5 min, 4°C).
- Membranes were collected from the supernatant fraction by centrifugation (32,000 x g, 18 min, 4°C), washed with ice-cold hypotonic buffer, and collected again by centrifugation (32,000 x g, 18 min, 4°C). The final membrane pellet was resuspended by sonication into a small volume of ice-cold binding buffer ( ⁇ 1 mL for every 5 plates: 10 mM NaCl, 20 mM HEPES, 0.22 mM KH 2 PO 4 , 1.26 mM CaCl 2 , 0.81 mM MgSO 4 , pH 7.4).
- Protein concentration was measured by the Bradford method (Bradford, 1976) using Bio-Rad Reagent, with bovine serum albumin as a standard. Membranes were held on ice for up to one hour and used fresh, or flash-frozen and stored in liquid nitrogen.
- Membranes were prepared similarly from 293, LM(tk-), and NlH-3T3 cells.
- To prepare membranes from baculovirus infected cells 2 x 10 7 Sf21 cells were grown in 150mm tissue culture dishes and infected with a high-titer stock of hY5BB3. Cells were incubated for 2-4 days at 27°C, no CO 2 before harvesting and membrane preparation as described above.
- Membranes were prepared similarly from dissected rat hypothalamus. Frozen hypothalami were homogenized for 20 seconds in ice-cold sonication buffer with the narrow probe of a Virtishear homogenizer at 1000 rpm (Virtis, Gardiner, NY). Large particles and debris were cleared by centrifugation (200 x g, 5 min, 4°C) and the supernatant fraction was reserved on ice. Membranes were further extracted from the pellet by repeating the homogenization and centrifugation procedure two more times. The supernatant fractions were pooled and subjected to high speed centrifugation (100,000 x g, 20 min. 4°C).
- 125 I-PYY (or alternative radioligand) and peptide competitors were also diluted to desired concentrations in supplemented binding buffer.
- Filter-trapped membranes were impregnated with MultiLex solid scintillant (Wallac, Turku, Finland) and counted for 125 I in a Wallac Beta-Plate Reader.
- Non-specific binding was defined by 300 nM human NPY for all receptors except the Y4 subtypes; 100 nM human PP was used for the human Y4 and 100 nM rat PP for the rat Y4.
- Specific binding in time course and competition studies was typically 80%; most non-specific binding was associated with the filter. Binding data were analyzed using nonlinear regression and statistical techniques available in the GraphPAD Prism package (San Diego, CA).
- the canine Y5 receptor pharmacology was investigated using porcine 125 I-PYY as described above. Nonspecific binding was defined by 1 ⁇ M human NPY. As above, membranes were collected by filtration over Whatman GF/C filters and counted for radioactivity.
- Stably transfected cells were seeded into 96-well microtiter plates and cultured until confluent. To reduce the potential for receptor desensitization, the serum component of the media was reduced to 1.5% for 4 to 16 hours before the assay. Cells were washed in Hank's buffered saline, or HBS (150 mM NaCl, 20 mM HEPES, 1 mM CaCl 2 , 5 mM KCl, 1 mM MgCl 2 , and 10 mM glucose) supplemented with 0.1% bovine serum albumin plus 5 mM theophylline and pre-equilibrated in the same solution for 20 min at 37°C in 5% CO 2 .
- HBS Hank's buffered saline
- HBS 150 mM NaCl, 20 mM HEPES, 1 mM CaCl 2 , 5 mM KCl, 1 mM MgCl 2 , and 10 mM glucose
- Intracellular calcium mobilization The intracellular free calcium concentration was measured by microspectroflourometry using the fluorescent indicator dye Fura-2/AM (ref). Stably transfected cells were seeded onto a 35 mm culture dish containing a glass coverslip insert. Cells were washed with HBS and loaded with 100 ⁇ l of Fura-2/AM (10 ⁇ M) for 20 to 40 min. After washing with HBS to remove the Fura-2/AM solution, cells were equilibrated in HBS for 10 to 20 min. Cells were then visualized under the 40X objective of a Leitz Fluovert FS microscope and fluorescence emission was determined at 510 nM with excitation wave lengths alternating between 340 nM and 380 nM. Raw fluorescence data were converted to calcium concentrations using standard calcium concentration curves and software analysis techniques.
- oligonucleotide probes employed to characterize the distribution of the rat NPY Y5 mRNA were complementary to nucleotides 1121 to 1165 in the 5,6-loop of the rat Y5 mRNA (Fig. 3A) 45mer antisense and sense oligonucleotide probes were synthesized on a Millipore Expedite 8909 Nucleic Acid Synthesis System. The probes were then lyophilized, reconstituted in sterile water, and purified on a 12% polyacrylamide denaturing gel. The purified probes were again reconstituted to a concentration of 100 ng/ ⁇ L, and stored at -20°C.
- Probes were 3'-end labeled with 35 S-dATP (1200 Ci/mmol, New England Nuclear, Boston, MA) to a specific activity of 10 9 dpm/ ⁇ g using terminal deoxynucleotidyl transferase (Pharmacia).
- the radiolabeled probes were purified on Biospin 6 chromatography columns (Bio-Rad; Richmond, CA), and diluted in hybridization buffer to a concentration of 1.5 x 10 4 cpm/ ⁇ L.
- One hundred ⁇ L of the diluted radiolabeled probe was applied to each section, which was then covered with a Parafilm coverslip. Hybridization was carried out overnight in humid chambers at 40 to 55°C.
- Controls for probe/hybridization specificity included hybridization with the radiolabeled sense probe, and the use of transfected cell lines. Briefly, COS-7 cells were transfected (see above) with receptor cDNAs for the rat Y1, Y2 (disclosed in US patent application 08/192,288, filed February 3, 1994), Y4 (disclosed in US patent application 08/176,412, filed December 28, 1993), or Y5. As described above, the transfected cells were treated and hybridized with the radiolabeled Y5 antisense and sense oligonucleotide probes, washed, and exposed to film for 1-7 days.
- Hybridization signal indicates the relative number of silver grains observed over neurons in a selected area of the rat brain.
- Two independent observers rated the intensity of the hybridization signal in a given brain area as nonexistent, low, moderate, or high. These were then converted to a subjective numerical scale as 0, +1, +2, or +3 (see Table 10), and mapped on to schematic diagrams of coronal sections through the rat brain (see Fig. 11).
- each process step is separated and/or isolated prior to its use as starting material for subsequent steps. Separation and isolation can be effect by any suitable purification procedure such as, for example, evaporation, crystallization, column chromatography, thin layer chromatography, distillation, etc. While preferred reactants have been identified herein, it is further contemplated that the present invention would include chemical equivalents to each reactant specifically enumerated in this disclosure.
- N,N-Dimethylaniline (114.0 g) is added slowly to a solution of 1H,3H-quinazolin-2,4-dione (146.0 g) in phosphorousoxychloride (535.4 ml) while this mixture is heated up to 140°C. After completion of the addition reflux is continued for 20 h. The reaction mixture is filtered and evaporated to give a residue which is added to ice and water. The product is extracted with dichloromethane and crystallized from diethylether and petroleum diethylether to yield 2,4-dichloro-quinazoline, m.p. 115 - 116°C.
- the starting material is prepared as follows:
- the reaction mixture is taken up in ice-cold dichloromethane, washed with an ice-cold 0.5 N HCl solution, a saturated aqueous sodium carbonate solution and water. The organics are dried over sodium sulfate and concentrated to 41.3 g of mixt- anhydride as an oil.
- This material is taken up in THF and treated at -70°C with sodium borohydride (5.90 g), followed by absolute methanol (10 ml) .
- the reaction mixture is stirred 15 h at 0°C and 1 h at ambient temperature to drive the reaction to completion.
- a 0.5N HCl solution is then carefully added at 0°C, followed by ethyl acetate.
- trans-(4-Hydroxymethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester (24 g) in pyridine (200 ml) at 0°C is treated with a solution of para-toluenesulfonylchloride (24.44 g) in pyridine (50 ml).
- the reaction mixture is stirred at 0°C until completion and concentrated in vacuo .
- the residue is taken up in ethyl acetate, washed with water and dried over sodium sulfate. Concentration of the solution yields the tosylate, used without further purification.
- trans-(4-Azidomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester (24 g) in ethyl acetate (1 liter) is hydrogenated over platinumoxide (2.4 g) at ambient temperature under atmospheric pressure of hydrogen.
- the catalyst is filtered-off and the filtrate concentrated to yield trans-(4-aminomethyl-cyclohexylmethyl)-carbamic acid tert-butyl ester as an oil.
- Rf(C2) 0.41.
- d) trans- ⁇ 4-[(Naphthalene-1-sulfonylamino)-methyl]- cyclohexylmethyl)-carbamic acid tert-butyl ester
- the starting material is prepared as follows:
- the starting material can be prepared as follows:
- Compounds of Formula 1-2 in Scheme 1 may be synthesized from the compound of Formula 1-1 by amidation using suitable methods such as those taught in "The Peptides,” Vol. 1 (Gross and Whyhofer, Eds. Acaemic Press, N.Y., 1979).
- the compound of Formula 1-1 may be treated with a carboxylic acid derivative of W in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and dimethylaminopyridine (DMAP) in a suitable solvent such as CH 2 Cl 2 as shown in Scheme 1, Step C, at room temperature in an inert atmosphere of argon or nitrogen, to yield the amide compound of Formula 1-2.
- EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
- DMAP dimethylaminopyridine
- the compound of Formula 1-2 may be synthesized by acylation of the amine of Formula 1-1 using the acid chloride of W, i.e., WCOCl, in a solvent such as CH 2 Cl 2 and a suitable tertiary amine such as triethylamine, at room temperature.
- W acid chloride of W
- a solvent such as CH 2 Cl 2
- a suitable tertiary amine such as triethylamine
- the product compounds of Formula 1-3 are then formed by reduction of the amide of Formula 1-3 using borane- tetrahydorfuran (THF) complex, in THF as shown in Scheme 1, Step D, at elevated temperature in an inert atmosphere.
- THF borane- tetrahydorfuran
- the compounds of Formula 3-1 may be deprotected using well known methods as shown in Scheme 2, Step C, and further sulfonylated with a sulfonyl halide of Ar, as shown in Scheme 2, Step D, in a suitable solvent such as CH 2 Cl 2 and a tertiary amine such as triethylamine, to form the compound of Formula 3-3.
- Compounds of Formula 3-3 may be reduced to yield the compounds of Formula 3-3, as shown in Scheme 2 , Step E, using borane-tetrahydorfuran (THF) complex, in THF, at elevated temperature in an inert atmosphere.
- THF borane-tetrahydorfuran
- Compound 25 was synthesized from the above compound by borane-THF reduction as follows:
- food intake may be measured in normal satiated rats after intracerebroventricular application (i.e.v.) of NPY in the presence or absence of the test compound.
- Male Sprague Dawley rats (Ciba-Geigy AG, Sisseln, Switzerland) weighing between 180g and 220g are used for all experiments. The rats are individually housed in stainless steel cages and maintained on an 11:13 h light-dark cycle (lights off at 18:00 h) at a controlled temperature of 21-23 °C at all times.
- Water and food (NAFAG lab chow pellets NAFAG, Gossau, Switzerland) are available ad libidum.
- Rats under pentobarbital anesthesia are stereotaxically implanted with a stainless steel guide cannula targeted at the right lateral ventricle.
- Stereotaxic coordinates, with the incisor bar set -2.0mm below interaural line, are: -0.8mm anterior and +1.3mm lateral to bregma.
- the guide cannula is placed on the dura.
- Injection cannulas extend the guide cannulas -3.8mm ventrally to the skull surface. Animals are allowed at least 4 days of recovery postoperatively before being used in the experiments.
- Cannula placement is checked postoperatively by testing all rats for their drinking response to a 50 ng intracerebroventricular (i.e. v.) injection of angiotensin II. Only rats which drink at least 2.5 ml of water within 30 min. after angiotensin II injection are used in the feeding studies.
- ACSF artificial cerebrospinal fluid
- p-NPY Porcine-NPY
- ACS artificial cerebrospinal fluid
- the test compounds are preferably dissolved in DMSO/water (10%, v/v).
- the vehicle used for intraperitoneal (i.p.), subcutaneous (s.c.) or oral (p.o.) delivery of compounds is preferably water, physiological saline or DMSO/water (10% v/v), or cremophor/water (20% v/v), respectively.
- NPY is administered by intracerebroventricular (i.e.v.) application.
- Food intake may be measured by placing preweighed pellets into the cages at the time of NPY injection. Pellets are then removed from the cage subsequently at each selected time point and replaced with a new set of preweighed pellets.
- the food intake of animals treated with test compound may be calculated as a percentage of the food intake of control animals i.e., animals treated with vehicle.
- food intake for each group of animals subjected to a particular experimental condition may be expressed as the mean ⁇ S.E.M.
- Statistical analysis is performed by analysis of variance using the Student-Newman-Keuls test.
- the animals After receipt, the animals are individually housed for the duration of the study and allowed free access to normal food together with tap water.
- the animals are maintained in a room with a 12 h light/dark cycle (8:00 a.m. to 8:00 p.m. light) at 24°C and monitored humidity.
- the rats undergo a 4 day equilibration period, during which they are habituated to their new environment and to eating a powdered or pellet diet NAFAG, Gossau, Switzerland).
- food is removed from the animals for 24 hours starting at 8:00 a.m.
- compound or vehicle may be administered to the animals orally or by injection intraperitoneally or intravenously.
- food is returned to the animals and their food intake is monitored at various time periods during the following 24 hour period.
- the food intake of animals treated with test compound may be calculated as a percentage of the food intake of control animals (i.e., animals treated with vehicle).
- food intake for each group of animals subjected to a particular experimental condition may be expressed as the mean ⁇ S.E.M.
- the antiobesity efficacy of the compounds according to the present invention might also be manifested in Zucker obese rats, which are known in the art as an animal model of obesity. These studies are conducted with male Zucker fatty rats (fa/fa Harlan CPB, Austerlitz NL) weighing between 480g and 500g. Animals are individually housed in metabolism cages for the duration of the study and allowed free access to normal powdered food and water. The animals are maintained in a room with a 12 h light/dark cycle (light from 8:00 A.M. to 8:00 P.M.) at 24°C and monitored humidity. After placement into the metabolism cages the rats undergo a 6 day equilibration period, during which they are habituated to their new environment and to eating a powdered diet.
- test compounds or vehicle preferably water or physiological saline or DMSO/water (10%, v/v) or cremophor/water (20%,v/v)
- Food intake is then monitored over the following 3 day period to determine the effect of administration of test compound or vehicle alone.
- food intake in the presence of drug may be expressed as a percentage of the food intake of animals treated with vehicle, or as the amount of food intake for a group of animals subjected to a particular experimental condition.
- Cell culture media and supplements are from Specialty Media (Lavallette, NJ).
- Cell culture plates 150 mm and 96-well microtiter) were from Corning (Corning, NY).
- Sf9, Sf21, and High Five insect cells, as well as the baculovirus transfer plasmid, pBlueBacIIITM, were purchased from Invitrogen (San Diego, CA).
- Ex-Cell 400TM medium with L- Glutamine was purchased from JRH Scientific.
- Polypropylene 96-well microtiter plates were from Co-star (Cambridge, MA). All radioligands were from New England Nuclear (Boston, MA). Commercially available NPY and related peptide analogs were either from Bachem California (Torrance, CA) or Peninsula (Belmont, CA); [D- Trp 32 ]NPY and PP C-terminal fragments were synthesized by custom order from Chiron Mimotopes Peptide Systems (San Diego, CA). Bio-Rad Reagent was from Bio-Rad (Hercules, CA). Bovine serum albumin (ultra-fat free, A-7511) was from Sigma (St. Louis. MO). All other materials were reagent grade.
- the competitive displacement data indicate: 1) Human PP is able to displace 20% of the bound 125 I-PYY with an IC 50 of 11 nM (Fig. 1 and Table 3). As can be seen in Table 5, this value does not fit with the isolated rat Y1, Y2 and Y4 clones and could therefore correspond to another NPY/PYY receptor subtype. 2) [Leu 31 , Pro 34 ] NPY (a Y1 specific ligand) is able to displace with high affinity (IC 50 of 0.38) 27% of the bound 125 I-PYY 3-36 ligand (a Y2 specific ligand) (Fig. 2 and Table 3).
- Binding data reflect competitive displacement of 125 I-PYY and 125 I-PYY 3-36 from rat hypothalamic membranes. Peptides were tested at concentrations ranging from 0.001 nM to 100 nM unless noted. The IC 50 value corresponding to 50% displacement, and the percentage of displacement relative to that produced by 300 nM human NPY, were determined by nonlinear regression analysis. Data shown are representative of at least two independent experiments.
- a rat hypothalamic cDNA library of 3 x 10 6 independent recombinants with a 2.7 kb average insert size was fractionated into 450 pools of ⁇ 7500 independent clones. All pools were tested in a binding assay with 125 I-PYY as previously described (US Serial No. 08/192,288). Seven pools gave rise to positive cells in the screening assay (#'s 81, 92, 147, 246, 254, 290, 312). Since Y1, Y2, Y4 and Y5 receptor subtypes (by PCR or binding analysis) are expressed in rat hypothalamus, the DNA of positive pools were analyzed by PCR with rat Y1, Y2 and Y4 specific primers.
- Pools # 147, 246, 254 and 31.2 turned out to contain cDNAs encoding a Y1 receptor; pool # 290 turned out to contain cDNA encoding a Y2 receptor subtype; but pools # 81 and 92 were negative by PCR analysis for Y1, Y2 and Y4 and therefore likely contained a cDNA encoding a new rat hypothalamic NPY receptor (Y5). Pools # 81 and 92 later turned out to contain an identical NPY receptor cDNA. Pool 92 was subjected to sib selection until a single clone was isolated (designated CG-18).
- the isolated clone carries a 2.8 kb cDNA.
- This cDNA contains an open reading frame between nucleotides 779 and 2146 that encodes a 456 amino acid protein.
- the long 5' untranslated region could be involved in the regulation of translation efficiency or mRNA stability.
- the flanking sequence around the putative initiation codon does not conform to the Kozak consensus sequence for optimal translation initiation (Kozak, 1989, 1991).
- the hydrophobicity plot displayed seven hydrophobic, putative membrane spanning regions which makes the rat hypothalamic Y5 receptor a member of the G-protein coupled superfamily.
- the nucleotide and deduced amino acid sequences are shown in Figures 3 and 4, respectively.
- the Y5 receptor contains consensus sequences for N-linked glycosylation, in the amino terminus (position 21 and 28) involved in the proper expression of membrane proteins (Kornfeld and Kornfeld, 1985).
- the Y5 receptor carries two highly conserved cysteine residues in the first two extracellular loops that are believed to form a disulfide bond stabilizing the functional protein structure (Probst et al, 1992).
- the Y5 receptor shows 9 potential phosphorylation sites for protein kinase C in positions 204, 217, 254, 273, 285, 301, 328, 336 and 409 and 2 cAMP- and cGMP-dependent protein kinase phosphorylation sites in positions 298 and 370.
- rat Y5 receptor carries a leucine zipper motif in its first putative transmembrane domain (Landschulz et al, 1988). A tyrosine kinase phosphorylation site is found in the middle of the leucine zipper.
- the isolated clone (CG- 19) turned out to contain a full length cDNA cloned in the correct orientation for functional expression (see below).
- the human Y5 nucleotide and deduced amino acid sequences are shown in Figures 5 and 6, respectively.
- the longest open reading frame encodes a 455 amino acid protein.
- the human sequence shows 84.1% nucleotide identity (Fig. 7A to 7E) and 87.2% amino acid identity (Fig. 7F and 7G).
- the rat protein sequence is one amino acid longer at the very end of both amino and carboxy tails of the receptor when compared to the human protein sequence.
- the human 5-6 loop is one amino acid longer than the rat and shows multiple non conservative substitutions.
- the longest open reading frame in the canine (beagle) Y5 cDNA (BOll) encodes a 456 amino acid protein with an estimated molecular weight of 50 kD.
- the full-length deduced canine Y5 amino acid sequence is shown in Figure 24.
- the canine Y5 receptor is the same length as the rat Y5 receptor, and is one amino acid longer than the human Y5 receptor.
- the canine Y5 receptor has 94.3% amino acid identity and 91.7% nucleotide identity with the human Y5 receptor.
- the canine Y5 receptor has 91.6% amino acid identity and 82.8% nucleotide identity with the rat Y5 receptor.
- Evidence was found for a potential allelic variation in the beagle Y5 receptor.
- the cDNA for the rat hypothalamic Y5 receptor was transiently expressed in COS-7 cells for full pharmacological evaluation.
- 125 I-PYY bound specifically to membranes from COS-7 cells transiently transfected with the rat Y5 receptor construct.
- the time course of specific binding was measured in the presence of 0.08 nM 125 l-PYY at 30°C (Fig. 9).
- the association curve was monophasic, with an observed association rate (K obs ) of 0.06 min -1 and a t 1/2 of 11 min; equilibrium binding was 99% complete within 71 min and stable for at least 180 min. All subsequent binding assays were carried out for 120 min at 30°C.
- the association curve in 10 mM [Na + ] was monophasic, with an observed association rate (K obs ) of 0.042 min -1 and a t 1/2 of 17 min; equilibrium binding was 99% complete within 110 min and stable for at least 210 min (specific binding was maximal at 480 fmol/mg membrane protein).
- the association curve in 138 mM [Na + ] was also monophasic with a slightly slower time course: (K obs ) of 0.029 min -1 and a t 1/2 of 24 min.; equilibrium binding was 99% complete within 160 min. and stable for at least 210 min. (specific binding was maximal at 330 fmol/mg membrane protein).
- the pharmacological profile of the rat Y5 receptor was first studied by using pancreatic polypeptide analogs in membrane binding assays. The rank order of affinity for selected compounds was derived from competitive displacement of 125 I-PYY (Fig. 11). The rat Y5 receptor was compared with cloned Y1, Y2, and Y4 receptors from human (Table 5) and rat (Table 6), all expressed transiently in COS-7 cells. One receptor subtype absent from our panel was the Y3, human or rat, as no model suitable for radioligand screening has yet been identified.
- Binding data reflect competitive displacement of 125 I-PYY from membranes of COS-7 cells transiently expressing rat Y5 and human subtype clones. Peptides were tested at concentrations ranging from 0.001 nM to 1000 nM unless noted. IC 50 values corresponding to 50% displacement were determined by nonlinear regression analysis and converted to K. values according to the Cheng-Prusoff equation. The data shown are representative of at least two independent experiments.
- Binding data reflect competitive displacement of 125 I-PYY from membranes of COS-7 cells transiently expressing rat Y5 and rat subtype clones. Peptides were tested at concentrations ranging from 0.001 nM to 1000 nM. IC 50 values corresponding to 50% displacement were determined by nonlinear regression analysis and converted to K i values according to the Cheng-Prusoff equation. The data shown are representative of at least two independent experiments. Exception: new peptides (marked with a double asterisk) were tested in one or more independent experiments.
- PYY and N-terminally deleted fragments such as PYY 3-36 and PYY 13-36 .
- This pattern places the binding profile of the Y5 receptor somewhere between that of the Y2 receptor (which receptor can withstand extreme N- terminal deletion) and that of the Y1 receptor (which receptor is sensitive to even a single-residue N-terminal deletion).
- the Y5 receptor resembled both Y1 and Y4 receptors in its tolerance for ligands containing Pro 34 (as in human [Leu 31 , Pro 34 ] NPY, human [Pro 34 ]-PYY, and human PP).
- K i 73 nM
- the rank order of K i values are in agreement with rank orders of potency and activity for stimulation of feeding behavior when peptides are injected i.e. v. or directly into rat hypothalamus (Clark et al., 1984; Stanley et al., 1985; Kalra et al., 1991; Stanley et al., 1992).
- the cDNA corresponding to the human Y5 homolog isolated from human hippocampus was transiently expressed in COS-7 cells for membrane binding studies.
- the binding of 125 I- PYY to the human Y5 receptor (CG-19) was saturable over a radioligand concentration range of 8 pM to 1.8 nM.
- a maximum receptor density of 500 fmol/mg membrane protein was measured on fresh membranes.
- the human Y5 pharmacological profile bears a striking resemblance to the rat Y5 receptor (Tables 7 and 8).
- Binding data reflect competitive displacement of radioligand (either 125 I-PYY or 125 I-PYY 3-36 as indicated) from membranes of COS-7 cells transiently expressing the rat Y5 receptor and its human homolog or from LM(tk-) cells stably expressing the human Y5 receptor.
- Peptides were tested at concentrations ranging from 0.001 nM to 1000 nM.
- IC 50 values corresponding to 50% displacement were determined by nonlinear regression analysis and converted to K i values according to the Cheng-Prusoff equation. New peptides are marked with a double asterisk.
- Binding data reflect competitive displacement of 125 I-PYY from membranes of COS-7 cells transiently expressing human Y5 other sub-type clones. Peptides were tested at concentrations ranging from 0.001 nM to 1000 nM unless noted. IC 50 values corresponding to 50% displacement were determined by nonlinear regression analysis and converted to K i values according to the Cheng-Prusoff equation. The data shown are representative of at least two independent experiments.
- Tests were initially performed to optimize expression of hY5 receptor. Infecting Sf9, Sf21, and High Five cells with hY5BB3 virus at a multiplicity of infection (MOI) of 5 and preparing membranes for binding analyses at 45 hrs. postinfection, B max ranges from 417 to 820 fmoles/mg protein, with the highest expression being hY5BB3 in Sf21 cells were observed. Therefore, the next series of experiments used Sf21 cells. Optimal multiplicity of infection (the ratio of viral particles to cells) was next examined by testing MOI of 1, 2, 5 and 10. The B values were ⁇ 1.1-1.2 pmoles/mg protein for any of the MOIs, suggesting that increasing the number of viral particles per cell is neither deleterious nor advantageous.
- MOI multiplicity of infection
- Sf21 cells infected with a human Y5 baculovirus construct were harvested as membrane homogenates and screened for specific binding of 125 I-PYY using 0.08 nM radioligand. Specific binding was greatest (500 fmol/mg membrane protein) for sample D-2/[4], derived from Sf-21 cells. No specific binding was observed after infection with the baculovirus plasmid alone (data not shown). If the assumption is made that the binding affinity of porcine 125 l-PYY for the human Y5 receptor is the same whether the expression system is COS-7 or baculovirus/Sf-21 (0.18 nM), the specific binding in sample D-2/[4] predicts an apparent B max of 1600 fmol/mg membrane protein. The Y5 receptor yield in the baculovirus/Sf21 expression system is therefore as good or better than that in COS-7. We conclude that the baculovirus offers an alternative transfection technique amenable to large batch production of the human Y5 receptor.
- Membranes from COS-7 cells transiently transfected with canine Y5 receptor displayed specific binding of porcine 125 I-PYY. The binding was saturable over a concentration range of 0.6 pM to 2.7 nM, with an observed K d of 1.1 nM and a B max of 5700 fmol/mg membrane protein.
- Compounds selected for the ability to bind or activate the human and rat Y5 receptor homologs were subsequently tested for binding to the canine Y5 receptor (Table 20). The pharmacological profile for the canine Y5 receptor was in general agreement with those derived for the other species homologs.
- the canine Y5 receptor exhibits what has been historically perceived as a Y1-like property.
- Y5-selective peptide D-[Trp 32 ]NPY and the Y1-selective antagonist BIBP 3226 were bound by the canine Y5 receptor with K i values of 35 and 17000 nM, respectively. These values are in the range of those reported for the rat and human Y5 homologs.
- BIBP 3226 was also tested for binding affinity at the cloned human Y-type receptors, and was observed to bind with K i values of 14 nM for the Y1 receptor, 6900 nM for the Y2 receptor, 8000 nM for the Y4 receptor and 49000 nM for the Y5 receptor. Similar experiments with cloned rat Y-type receptors generated K i values of 20 nM for the Y1 receptor, 66000 nM for the Y2 receptor, 420 nM for the Y4 receptor and 25000 nM for the Y5 receptor.
- BIBP 3226 blocked NPY-induced activation of rat Y1 receptors with a K D of 9.4 nM and also blocked PP-induced activation of rat Y4 receptors with a Kb of 4800 uM; there was no evidence for antagonism of NPY- or PP-induced activation of rat Y2 or Y5 receptors at concentrations up to 1 ⁇ M. These data further confirm the classification of BIBP 3226 as a Y1-selective receptor antagonist. Stable Expression Systems for Y5 Receptors: Characterization in Binding Assays
- the cDNA for the rat Y5 receptor was stably transfected into 293 cells which were pre-screened for the absence of specific 125 I-PYY binding (data not shown). After co- transfection with the rat Y5 cDNA plus a G-418-resistance gene and selection with G-418, surviving colonies were screened as membrane homogenates for specific binding of 125 I-PYY using 0.08 nM radioligand. A selected clone (293 clone # 12) bound 65 fmol 125 I-PYY /mg membrane protein and was isolated for further study in functional assays.
- the cDNA for the human Y5 receptor was stably transfected into both NlH-3T3 and LM(tk-) cells, each of which were pre-screened for the absence of specific 125 l-PYY binding (data not shown). After co-transfection with the human Y5 cDNA plus a G-418-resistance gene and selection with G- 418, surviving colonies were screened as membrane homogenates for specific binding of 125 I-PYY using 0.08 nM radioligand. N1H-3T3 clone #8 bound 46 fmol 125 I-PYY/mg membrane protein and LM(tk-) clone #7 bound 32 fmol 125 I- PYY/mg membrane protein.
- the human Y5 stably expressed in LM(tk-) cells was further characterized in saturation binding assays using 125 I-PYY, 125 I-PYY 3-36 , and 125 I-NPY.
- Peptide K. values derived from 125 I-PYY binding to human Y5 receptors from LM(tk-) were comparable to those derived from the previously described human and rat Y5 expression systems
- 125 I-NPY binding to the human Y5 in LM(tk-) cells was saturable according to a l-site model over a concentration range of 0.4 pM to 1.19 nM, with an apparent K d of 0.28 and an apparent B max of 360 fmol/mg membrane protein when membranes had been frozen and stored in liquid nitrogen.
- the data provide evidence that the Y5 receptor is a target for multiple radioiodinated peptide analogs in the pancreatic polypeptide family, including 125 I-PYY, 125 I-NPY, 125 I-PYY 3-36 , and 125 I-[Leu 31 ,Pro 34 ]PYY.
- the so-called Y1 and Y2- selective radioligands should be used with caution when probing native tissues for Y-type receptor expression.
- a portion of the receptor population can typically be characterized in the high affinity ligand binding site using discriminating agonists.
- the binding of GTP or a non-hydrolyzable analog to the G protein causes a conformational change in the receptor which favors a low affinity ligand binding state.
- Gpp(NH)p would alter the binding of 125 I-PYY to Y5 in COS- 7 and LM(tk-) cells (Fig 19) was investigated.
- the difference between the receptor preparations could be explained by several factors, including 1) the types of G proteins available in the host cell for supporting a high affinity receptor- agonist complex, 2) the level of receptor reserve in the host cell, 3) the efficiency of receptor/G protein coupling, and 4) the intrinsic ability of the agonist (in this case, 125 I-PYY) to distinguish between multiple conformations of the receptor.
- Activation of all Y-type receptors described thus far is thought to involve coupling to pertussis toxin-sensitive G-proteins which are inhibitory for adenylate cyclase activity (G i or G o ) (Wahlestedt and Reis, 1993). That the atypical Y1 receptor is linked to cyclase inhibition was prompted by the observation that pertussis toxin inhibited NPY-induced feeding in vivo (Chance et al., 1989); a more definitive analysis was impossible in the absence of the isolated receptor.
- IC values derived from rat Y5-dependent binding of 125 I-PYY and peptide ligands were in close range of EC 50 values derived from rat Y5-dependent regulation of cAMP accumulation (Table 9).
- the maximal suppression of cAMP produced by all peptides in Table 9 was between 84% and 120% of that produced by human NPY, except in the case of FLRFamide (42%).
- the Y5-selective peptide [D-Trp 32 ]NPY is a peptide which was shown to stimulate food intake when injected into rat hypothalamus, and which also attenuated NPY-induced feeding in the same paradigm (Balasubramaniam, 1994).
- [D- Trp 32 ]NPY bound weakly to other Y-type clones with K i > 500 nM (Tables 5 and 6) and displayed no activity in functional assays (Table 11).
- Functional data were derived from radioimmunoassay of cAMP accumulation in stably transfected 293 cells stimulated with 10 ⁇ M forskolin. Peptides were tested for agonist activity at concentrations ranging from 0.03 pM to 0.3 ⁇ M. The maximum inhibition of cAMP accumulation (E max ) and the concentration producing a half- maximal effect (EC 50 ) were determined by nonlinear regression analysis according to a 4 parameter logistic equation. New peptides are marked with a double asterisk.
- the human Y5 receptor supported a cellular response to NPY-like peptides in a rank order similar to that described for the rat Y5 receptor (Table 6, 10).
- the human Y5 receptor is clearly linked by [D- Trp 32 ]NPY and other pharmacological tools to the NPY- dependent regulation of feeding behavior, the human Y5 receptor is predicted to function in a similar fashion.
- Both the human and receptor homologs represent useful models for the screening of compounds intended to modulate feeding behavior by interfering with NPY- dependent pathways. TABLE 10: Functional activation of the human Y5 receptor in a cAMP radioimmunoassay.
- Functional data were derived from radioimmunoassay of cAMP accumulation in stably transfected LM(tk-) cells stimulated with 10 ⁇ M forskolin. Peptides were tested for agonist activity at concentrations ranging from 0.03 pM to 0.3 ⁇ M. The maximum inhibition of cAMP accumulation (E max ) and the concentration producing a half- maximal effect (EC 50 ) were determined by nonlinear regression analysis according to a 4 parameter logistic equation.
- Trp 32 Trp 32 ]NPY.
- Binding data were generated as described in Tables 5 and 6. Functional data were derived from radioimmunoassay of cAMP accumulation in stably transfected cells stimulated with 10 ⁇ M forskolin.
- [D-Trp 32 ]NPY was tested for agonist activity at concentrations ranging from 0.03 pM to 0.3 ⁇ M.
- [D-Trp 32 ]NPY was included as a single spike (0.3 ⁇ M) in the human PYY concentration curve for human Y1 and human Y2 receptors, or in the human PP concentration curve for human Y4 receptors, and antagonist activity was detected by the presence of a rightward shift (from EC 50 to EC 50 ').
- Intracellular Calcium Mobilization Intracellular Calcium Mobilization
- Untransfected LM(tk-) cells did not respond to human NPY (data not shown).
- the calcium mobilization provides a second pathway through which Y5 receptor activation can be measured.
- These data also serve to link with the Y5 receptor with other cloned human Y-type receptors, all of which have been demonstrated to mobilize intracellular calcium in various expression systems (Fig 21).
- the mRNA for the NPY Y5 receptor was widely distributed in rat brain, and appeared to be moderately abundant (Table 12 and Fig. 13).
- the midline thalamus contained many neurons with silver grains over them, particularly the paraventricular thalamic nucleus, the rhomboid nucleus, and the nucleus reunions.
- moderately intense hybridization signals were observed over neurons in both the centromedial and anterodorsal thalamic nuclei.
- a moderate level of hybridization signal was seen over scattered neurons in the lateral hypothalamus, paraventricular, supraoptic, arcuate, and dorsomedial nuclei.
- the most intense signals were found over neurons in the anterior and olivary pretectal nuclei, periaquaductal gray, and over the rostral linear raphe. Moderate hybridization signals were observed over neurons in the internal gray layer of the superior colliculus, the substantia nigra, pars compacta, the dorsal raphe, and the pontine nuclei. Most of the neurons in the inferior colliculus exhibited a low level of signal. In the medulla and pons, few areas exhibited substantial hybridization signals. The medial vestibular nucleus was moderately labeled, as was the parvieellular reticular nucleus, pars alpha, and the gigantocellular reticular nucleus.
- Table 14 discloses several compounds which bind selectively to the human Y5 receptor and act as Y5 receptor antagonists, as measured by their ability to block NPY-induced inhibition of cAMP accumulation in forskolin-stimulated LM(tk-) cells stably transfected with the cloned human Y5 receptor.
- the structures of the compounds described in Table 13 are shown in Figure 22. Preliminary experiments indicate that compound 28 is a Y5 receptor antagonist.
- Table 14 Evaluation of human Y5 receptor antagonists
- K b The ability of the compounds to antagonize the Y-type receptors is reported as the K b .
- NPY-induced food intake was significantly reduced in animals first treated with the compounds (p ⁇ 0.05; Student-Newman-Keuls). These experiments demonstrate that NPY-induced food intake is significantly reduced by administration to animals of a compound which is a Y5-selective antagonist. Table 14 continued
- the Y5 antagonists of Table 14 were administered by intraperitoneal injection at a dose of 30 mg/kg to conscious rats following a 24h food deprivation.
- the human Y5 receptor antagonists shown in Table 14 reduced food intake in the food-deprived animals, as shown below in Table 16.
- the food intake of animals treated with test compound is reported as the percentage of the food intake measured for control animals (treated with vehicle), i.e., 25% means the animals treated with the compound consumed only 25% as much food as the control animals. Measurements were performed two hours after administration of the test compound.
- the longest reading frame in the rat Y5 cDNA encodes a 456 amino acid protein with an estimated molecular weight of 50.1 kD. Given there are two N-linked glycosylation sites in the amino terminus, the apparent molecular weight could be slightly higher.
- the human Y5 homolog was isolated from a human hippocampal cDNA library.
- the longest reading frame in the human Y5 cDNA encodes a 455 amino acid protein with an estimated molecular weight of 50 kD.
- the human Y5 receptor is one amino acid shorter than the rat Y5 and shows significant amino acid differences both in the N-terminal and the middle of the third intracellular loop portions of the protein.
- the rat and human Y5 receptors both carry a leucine zipper in the first putative transmembrane domain.
- segments containing periodic arrays of leucine residues exist in an alpha-helical conformation.
- the leucine side chains extending from one alpha-helix interact with those from a similar alpha helix of a second polypeptide, facilitating dimerization by the formation of a coiled coil (O'Shea et al, 1989) .
- Y5 rat and human
- CG-18 and CG-19 are named "Y5" receptors because of their unique amino acid sequence (87.2% identical with each other, ⁇ 42% identical with the TM regions of previously cloned "Y" receptor subtypes) and pharmacological profile. The name is not biased toward any one member of the pancreatic polypeptide family.
- the number is the next available in the Y-type series, position number three having been reserved for the pharmacologically defined Y3 receptor.
- the cloned human Y1 receptor was introduced by Larhammar and co-workers as a "human neuropeptide Y/peptide YY receptor of the Y1 type" (Larhammar et al., 1992).
- the novel clones described herein can be described as rat, human and canine neuropeptide Y/peptide YY receptors of the Y5 type.
- GenBank database An electronic search of the GenBank database for sequences with similarity to the human Y5 receptor sequences identified a match between the reverse complement of the human Y5 coding sequence and the human Y1 receptor exon IC and its flanking sequences. Exon IC is located in the 5'-untranslated region of the Y1C alternate splice variant mRNA of the human Y1 receptor (Ball, et al., 1995). This data reveals that the human Y1 and Y5 receptor genes map, in opposite orientation, to the same locus on chromosome 4q (see Figure 25).
- the rat hypothalamic Y5 receptor displays a very similar pharmacological profile to the pharmacologically described "atypical" Y1 receptor thought to mediate NPY-induced food intake in rat hypothalamus.
- Both the Y5 receptor and the "feeding receptor” display a preference for NPY and PYY-like analogs, a sensitivity to N-terminal peptide deletion, and a tolerance for Pro 34 .
- PP ligands which are capable of activating the Y5 receptor with high potency, such as bovine and human PP, contain a proline in position 13 or 14. While this proline is conserved in several PP ligands (porcine, sheep, and canine, for example) and also in human and porcine NPY as well as human and porcine PYY, it is not conserved in rat PP. This structural difference may lead to changes in protein folding and ultimately to changes in receptor interaction which underlie the relatively poor potency of rat PP for Y5 receptor activation. The understanding of these structure-activity relationships may be important for the design of Y5 selective ligands with the ability to modulate food intake in vivo .
- the distribution of Y5 mRNA in rat brain further extends the argument for a role of Y5 receptors in feeding behavior.
- the anatomical locus of the feeding response for example, has been suggested to reside at least in part in the paraventricular hypothalamic nucleus (PVN) and also in the lateral hypothalamus, two places where Y5 mRNA was detected in abundance. Post- synaptic localization of the Y5 receptor in both of these regions can regulate the response to endogenously released NPY in vivo.
- the paraventricular nucleus receives projections from NPY-containing neurons in the arcuate nucleus, another region where Y5 mRNA was detected.
- Y5 receptor This indicates a pre-synaptic role for the Y5 receptor in the control of NPY release via the arcuato-paraventricular projection, and consequently in the control of feeding behavior.
- the localization of the Y5 mRNA in the midline thalamic nuclei is also important.
- the paraventricular thalamic nucleus/centromedial nucleus complex projects heavily to the paraventricular hypothalamus and to the amygdala.
- the Y5 receptor is a substrate for the emotional aspect of appetitive behaviors.
- NPY neuropeptide Y5 receptors
- NPY is the most potent stimulant of feeding behavior yet described (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984).
- Direct injection of NPY into the hypothalamus of rats can increase food intake ⁇ 10-fold over a 4-hour period (Stanley et al., 1992).
- NPY- stimulated rats display a preference for carbohydrates over protein and fat (Stanley et al., 1985).
- NPY and NPY mRNA are increased in food- deprived rats (Brady et al., 1990; 0' Shea and Gundlach, 1991) and also in rats which are genetically obese (Sanacora et al., 1990) or made diabetic by treatment with streptozotocin (White et al., 1990).
- NPY a potent stimulant of feeding behavior in normal rats, is disregulated in the overweight or diabetic animal so that food intake is increased, accompanied by obesity.
- the physiological stress of obesity increases the risk for health problems such as cardiovascular malfunction, osteoarthritis, and hyperinsulinemia, together with a worsened prognosis for adult-onset diabetes.
- a nonpeptide antagonist targeted to the Y5 receptor could therefore be effective as a way to control not only appetite and body weight but an entire range of obesity- and diabetes-related disorders (Dryden et al., 1994).
- NPY-mediated functions are disregulated in eating disorders such as bulimia and anorexia nervosa, so that they too could be responsive to treatment by a Y5- selective drug. It has been proposed, for example, that food intake in NPY-stimulated rats mimics the massive food consumption associated with binge eating in bulimia (Stanley, 1993).
- Cerebro-spinal fluid (CSF) levels of PYY but not NPY were elevated in bulimic patients who abstained from binging, and then diminished when binging was allowed (Berrettini et al., 1988). Conversely, NPY levels were elevated in underweight anorectic patients and then diminished as body weight was normalized (Kaye et al., 1990).
- NPY is highly conserved across species (e.g. 100% in human, rat, guinea pig, rabbit and alligator) such that canine NPY is predicted to resemble human NPY, although the sequence of canine NPY is currently unknown.
- Canine and human PYY differ in only 2 out of 36 positions, whereas canine PYY is identical to porcine PYY.
- the canine Y5 receptor appears to be a plausible target not only for NPY synthesized in the canine nervous system, but also for circulating or neurally-derived PYY and PP.
- the canine Y5 receptor mediates all of the functions proposed for human and rat Y5 receptors, including the stimulation of feeding behavior.
- the cloned canine Y5 receptor and canine in vivo models are therefore believed to comprise a useful system with which to evaluate biological actions of Y5-selective compounds for the treatment of obesity and eating disorders in humans.
- the Y5 pharmacological profile further offers a new standard by which to review the molecular basis of all NPY-dependent processes; examples are listed in Table 18. Such an exercise suggests that the Y5 receptor is likely to have a physiological significance beyond feeding behavior. It has been reported, for example, that a Y-type receptor can regulate luteinizing hormone releasing hormone (LHRH) release from the median eminence of steroid-primed rats in vitro with an atypical Y1 pharmacological profile.
- LHRH luteinizing hormone releasing hormone
- NPY, NPY 2-36 , and LP-NPY were all effective at luM but deletion of as few as four amino acids from the N-terminus of NPY destroyed biological activity. The Y5 may therefore represent a therapeutic target for sexual or reproductive disorders.
- a successful strategy for the design of a Y5-receptor based drug or for any drug targeted to single G protein-coupled receptor subtype involves the screening of candidate compounds 1) in radioligand binding assays so as to detect affinity for cross-reactive G protein- coupled receptors, and 2) in physiological assays so as to detect undesirable side effects.
- the receptor subtypes most likely to cross-react and therefore most important for radioligand binding screens include the other "Y-type" receptors, Y1, Y2, Y3, and Y4.
- Cross-reactivity between the Y5 and any of the other subtypes could result in potential complications as suggested by the pathophysiological indications listed in Table 18.
- the cloning of the Y5 receptor from human and rat is especially valuable for receptor characterization based on in situ localization, anti-sense functional knockout, and gene induction. These studies will generate important information related to Y5 receptor function and its therapeutic significance.
- the cloned Y5 receptor lends itself to mutagenesis studies in which receptor/ligand interactions can be modeled.
- the Y5 receptor further allows us to investigate the possibility of other Y-type receptors through homology cloning. These could include new receptor subtypes as well as Y5 species homologs for the establishment of experimental animal models with relevance for human pathology.
- the Y5 receptor therefore represents an enormous opportunity for the development of novel and selective drug therapies, particularly those targeted to appetite and weight control, but also for memory loss, depression, anxiety, gastric ulcer, epileptic seizure, pain, hypertension, subarachnoid hemorrhage, sleeping disturbances, nasal congestion, neurogenic voiding dysfuncion, and diarrhea.
- Y5-selective antagonists which inhibit food intake in rats provides a method of modifying feeding behavior in a wide variety of vertebrate animals.
- K i IC 50 /(1 + [L]/K d ).
- n 2.
- n 3.
- Neuropeptide Y vasoconstrictor effects and possible role in cerebral vasospasm after experimental subarachnoid hemorrhage. Brain Res. 463: 250-258.
- Neuropeptide Y Role in light-dark cycle entrainment of hamster circadian rhythms. Neurosci. Lett. 50: 163-168.
- Neuropeptide Y A specific Y 1 receptor agonist. Proc. Natl. Acad. Sci. USA 87: 182-186.
- the 5-HT 4 receptor molecular cloning and pharmacological characterization of two splice variants. EMBO J. 14 (12):2806-2815.
- Neuropeptide Y infusion improves hemodynamics and survival in rat endotoxic shock. Am. J. Physiol. 265: H1416-H1423. Heilig, M., and Widerlov, E. (1990). Neuropeptide Y: an overview of central distribution, functional aspects, and possible involvement in neuropsychiatric illnesses. Acta Psychiatr. Scand. 82: 95-114.
- Neuropeptide Y A potent inducer of consummatory behavior in rats. Peptides 5: 1025-1029.
- Pancreatic polypeptide A possible role in the regulation of food intake in the mouse. Hypothesis. Experientia 33: 915- 917. McCormick, M. (1987). Sib Selection. Methods in Enzymology, 151: 445-449.
- Neuropeptide Y Stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci. 35: 2635-2642.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Neurology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP97928786A EP1007073A4 (en) | 1996-06-04 | 1997-06-04 | Methods of modifying feeding behavior, compounds useful in such methods, and dna encoding a hypothalamic atypical neuropeptide y/peptide yy receptor (y5) |
AU32952/97A AU3295297A (en) | 1996-06-04 | 1997-06-04 | Methods of modifying feeding behavior, compounds useful in such methods, and dna encoding a hypothalamic atypical neuropeptide y/peptide yy receptor (y5) |
US09/194,895 US6713265B1 (en) | 1997-06-04 | 1997-06-04 | Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/668,650 | 1996-06-04 | ||
US08/668,650 US5989920A (en) | 1994-12-02 | 1996-06-04 | Methods of modifying feeding behavior compounds useful in such methods and DNA encoding a hypothalmic atypical neuropeptide Y/peptide YY receptor Y5 |
US80360097A | 1997-02-21 | 1997-02-21 | |
US08/803,600 | 1997-02-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1997046250A1 true WO1997046250A1 (en) | 1997-12-11 |
Family
ID=27099966
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1997/009504 WO1997046250A1 (en) | 1996-06-04 | 1997-06-04 | Methods of modifying feeding behavior, compounds useful in such methods, and dna encoding a hypothalamic atypical neuropeptide y/peptide yy receptor (y5) |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1007073A4 (en) |
AU (1) | AU3295297A (en) |
WO (1) | WO1997046250A1 (en) |
Cited By (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0862627A1 (en) * | 1995-11-09 | 1998-09-09 | Garvan Institute Of Medical Research | Neuropeptide y-y5 receptor |
US6127414A (en) * | 1997-09-23 | 2000-10-03 | Astra Aktiebolag | NPY antagonists |
US6191160B1 (en) | 1998-11-10 | 2001-02-20 | Merck & Co., Inc. | Spiro-indolines as Y5 receptor antagonists |
US6645774B1 (en) | 1994-12-02 | 2003-11-11 | Synaptic Pharmaceutical Corporation | Methods of modifying feeding behavior using compounds with afinity for the human hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
US6713265B1 (en) | 1997-06-04 | 2004-03-30 | Synaptic Pharmaceutical Corporation | Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
US6713473B1 (en) | 1999-04-20 | 2004-03-30 | Meiji Seika Kaisha, Ltd. | Tricyclic compounds |
WO2004066966A2 (en) | 2003-01-17 | 2004-08-12 | Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. | Peptide yy analogs |
JP2004315511A (en) * | 2003-03-31 | 2004-11-11 | Taisho Pharmaceut Co Ltd | Mch receptor antagonist |
US6818445B2 (en) | 1994-12-02 | 2004-11-16 | Synaptic Pharmaceutical Corporation | Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
WO2004098591A2 (en) | 2003-05-05 | 2004-11-18 | Probiodrug Ag | Inhibitors of glutaminyl cyclase and their use in the treatment of neurological diseases |
WO2005049027A2 (en) | 2003-11-03 | 2005-06-02 | Probiodrug Ag | Combinations useful for the treatment of neuronal disorders |
WO2005075436A2 (en) | 2004-02-05 | 2005-08-18 | Probiodrug Ag | Novel inhibitors of glutaminyl cyclase |
US7034034B2 (en) | 2001-10-23 | 2006-04-25 | Neurogen Corporation | Substituted 2-cyclohexyl-4-phenyl-1H-imidazole derivatives |
WO2008055945A1 (en) | 2006-11-09 | 2008-05-15 | Probiodrug Ag | 3-hydr0xy-1,5-dihydr0-pyrr0l-2-one derivatives as inhibitors of glutaminyl cyclase for the treatment of ulcer, cancer and other diseases |
WO2008065141A1 (en) | 2006-11-30 | 2008-06-05 | Probiodrug Ag | Novel inhibitors of glutaminyl cyclase |
WO2008104580A1 (en) | 2007-03-01 | 2008-09-04 | Probiodrug Ag | New use of glutaminyl cyclase inhibitors |
US7550489B2 (en) | 2002-03-12 | 2009-06-23 | Merck & Co., Inc. | Substituted pyridyoxy amides |
US7589197B2 (en) | 2005-06-14 | 2009-09-15 | Taigen Biotechnology | Pyrimidine compounds |
WO2009146539A1 (en) * | 2008-06-02 | 2009-12-10 | Neuromed Pharmaceuticals Ltd. | 4-(aminomethyl)cyclohexanamine derivatives as calcium channel blockers |
WO2011029920A1 (en) | 2009-09-11 | 2011-03-17 | Probiodrug Ag | Heterocylcic derivatives as inhibitors of glutaminyl cyclase |
WO2011107530A2 (en) | 2010-03-03 | 2011-09-09 | Probiodrug Ag | Novel inhibitors |
WO2011110613A1 (en) | 2010-03-10 | 2011-09-15 | Probiodrug Ag | Heterocyclic inhibitors of glutaminyl cyclase (qc, ec 2.3.2.5) |
WO2011131748A2 (en) | 2010-04-21 | 2011-10-27 | Probiodrug Ag | Novel inhibitors |
US8193206B2 (en) | 2005-06-14 | 2012-06-05 | Taigen Biotechnology Co., Ltd. | Pyrimidine compounds |
WO2012123563A1 (en) | 2011-03-16 | 2012-09-20 | Probiodrug Ag | Benz imidazole derivatives as inhibitors of glutaminyl cyclase |
US8372849B2 (en) | 2008-04-21 | 2013-02-12 | Taigen Biotechnology Co., Ltd. | Heterocyclic compounds |
EP2865670A1 (en) | 2007-04-18 | 2015-04-29 | Probiodrug AG | Thiourea derivatives as glutaminyl cyclase inhibitors |
US9023834B2 (en) | 2008-11-13 | 2015-05-05 | Taigen Biotechnology Co., Ltd. | Lyophilization formulation |
EP3461819A1 (en) | 2017-09-29 | 2019-04-03 | Probiodrug AG | Inhibitors of glutaminyl cyclase |
US11427540B2 (en) | 2019-07-11 | 2022-08-30 | Praxis Precision Medicines, Inc. | Formulations of T-type calcium channel modulators and methods of use thereof |
US12077502B2 (en) | 2022-07-22 | 2024-09-03 | Praxis Precision Medicines, Inc. | Formulations of T-type calcium channel modulators and methods of use thereof |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5602024A (en) * | 1994-12-02 | 1997-02-11 | Synaptic Pharmaceutical Corporation | DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) and uses thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AUPN646795A0 (en) * | 1995-11-09 | 1995-11-30 | Garvan Institute Of Medical Research | Neuropeptide Y-Y5 receptor |
AU7626496A (en) * | 1995-12-01 | 1997-06-27 | Ciba-Geigy Ag | Heteroaryl compounds |
WO1997020822A1 (en) * | 1995-12-01 | 1997-06-12 | Novartis Ag | Quinazolin-2,4-diazirines as npy receptor antagonist |
US5965392A (en) * | 1996-04-08 | 1999-10-12 | Bayer Corporation | Neuropeptide Y receptor Y5 and nucleic acid sequences |
-
1997
- 1997-06-04 EP EP97928786A patent/EP1007073A4/en not_active Withdrawn
- 1997-06-04 AU AU32952/97A patent/AU3295297A/en not_active Abandoned
- 1997-06-04 WO PCT/US1997/009504 patent/WO1997046250A1/en not_active Application Discontinuation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5602024A (en) * | 1994-12-02 | 1997-02-11 | Synaptic Pharmaceutical Corporation | DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) and uses thereof |
Non-Patent Citations (4)
Title |
---|
GEHLERT D.R.: "SUBTYPES OF RECEPTORS FOR NEUROPEPTIDE Y: IMPLICATIONS FOR THE TARGETING OF THERAPEUTICS.", LIFE SCIENCES., PERGAMON PRESS, OXFORD, GB, vol. 55., no. 08., 1 January 1994 (1994-01-01), GB, pages 551 - 562., XP000612039, ISSN: 0024-3205, DOI: 10.1016/0024-3205(94)00481-1 * |
HERZOG H ET AL: "CLONED HUMAN NEUROPEPTIDE Y RECEPTOR COUPLES TO TWO DIFFERENT SECOND MESSENGER SYSTEMS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 89, no. 13, 1 January 1992 (1992-01-01), US, pages 5794 - 5798, XP002180040, ISSN: 0027-8424, DOI: 10.1073/pnas.89.13.5794 * |
See also references of EP1007073A4 * |
WAHLESTEDT C, REGUNATHAN S, REIS D J: "IDENTIFICATION OF CULTURED CELLS SELECTIVELY EXPRESSING Y1-, Y2-, OR Y3-TYPE RECEPTORS FOR NEUROPEPTIDE Y/PEPTIDE YY", LIFE SCIENCES., PERGAMON PRESS, OXFORD, GB, vol. 50, no. 05, 1 January 1991 (1991-01-01), GB, pages PL07 - PL12, XP001059170, ISSN: 0024-3205 * |
Cited By (42)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6645774B1 (en) | 1994-12-02 | 2003-11-11 | Synaptic Pharmaceutical Corporation | Methods of modifying feeding behavior using compounds with afinity for the human hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
US6818445B2 (en) | 1994-12-02 | 2004-11-16 | Synaptic Pharmaceutical Corporation | Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
EP0862627A4 (en) * | 1995-11-09 | 2000-01-12 | Garvan Inst Med Res | Neuropeptide y-y5 receptor |
EP0862627A1 (en) * | 1995-11-09 | 1998-09-09 | Garvan Institute Of Medical Research | Neuropeptide y-y5 receptor |
US6528303B1 (en) | 1995-11-09 | 2003-03-04 | Garvan Institute Of Medical Research | Neuropeptide Y-Y5 receptor |
US6713265B1 (en) | 1997-06-04 | 2004-03-30 | Synaptic Pharmaceutical Corporation | Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
US6127414A (en) * | 1997-09-23 | 2000-10-03 | Astra Aktiebolag | NPY antagonists |
US6313298B1 (en) | 1998-11-10 | 2001-11-06 | Merck & Co., Inc. | Spiro-indolines as Y5 receptor antagonists |
US6638942B1 (en) | 1998-11-10 | 2003-10-28 | Merck & Co., Inc. | Spiro-indolines as Y5 receptor antagonists |
US6495559B2 (en) | 1998-11-10 | 2002-12-17 | Merck & Co., Inc. | NPY Y5 receptor antagonists |
US6191160B1 (en) | 1998-11-10 | 2001-02-20 | Merck & Co., Inc. | Spiro-indolines as Y5 receptor antagonists |
US6713473B1 (en) | 1999-04-20 | 2004-03-30 | Meiji Seika Kaisha, Ltd. | Tricyclic compounds |
US7034034B2 (en) | 2001-10-23 | 2006-04-25 | Neurogen Corporation | Substituted 2-cyclohexyl-4-phenyl-1H-imidazole derivatives |
US7816534B2 (en) | 2002-03-12 | 2010-10-19 | Merck Sharp & Dohme Corp. | Substituted amides |
US7550489B2 (en) | 2002-03-12 | 2009-06-23 | Merck & Co., Inc. | Substituted pyridyoxy amides |
WO2004066966A2 (en) | 2003-01-17 | 2004-08-12 | Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. | Peptide yy analogs |
EP2277527A2 (en) | 2003-01-17 | 2011-01-26 | Ipsen Pharma | Peptide YY analogs |
US7811989B2 (en) | 2003-01-17 | 2010-10-12 | Ipsen Pharma S.A.S. | Peptide YY analogs |
JP2004315511A (en) * | 2003-03-31 | 2004-11-11 | Taisho Pharmaceut Co Ltd | Mch receptor antagonist |
WO2004098591A2 (en) | 2003-05-05 | 2004-11-18 | Probiodrug Ag | Inhibitors of glutaminyl cyclase and their use in the treatment of neurological diseases |
WO2005049027A2 (en) | 2003-11-03 | 2005-06-02 | Probiodrug Ag | Combinations useful for the treatment of neuronal disorders |
EP2338490A2 (en) | 2003-11-03 | 2011-06-29 | Probiodrug AG | Combinations Useful for the Treatment of Neuronal Disorders |
WO2005075436A2 (en) | 2004-02-05 | 2005-08-18 | Probiodrug Ag | Novel inhibitors of glutaminyl cyclase |
US7589197B2 (en) | 2005-06-14 | 2009-09-15 | Taigen Biotechnology | Pyrimidine compounds |
US8193206B2 (en) | 2005-06-14 | 2012-06-05 | Taigen Biotechnology Co., Ltd. | Pyrimidine compounds |
WO2008055945A1 (en) | 2006-11-09 | 2008-05-15 | Probiodrug Ag | 3-hydr0xy-1,5-dihydr0-pyrr0l-2-one derivatives as inhibitors of glutaminyl cyclase for the treatment of ulcer, cancer and other diseases |
WO2008065141A1 (en) | 2006-11-30 | 2008-06-05 | Probiodrug Ag | Novel inhibitors of glutaminyl cyclase |
WO2008104580A1 (en) | 2007-03-01 | 2008-09-04 | Probiodrug Ag | New use of glutaminyl cyclase inhibitors |
EP2481408A2 (en) | 2007-03-01 | 2012-08-01 | Probiodrug AG | New use of glutaminyl cyclase inhibitors |
EP2865670A1 (en) | 2007-04-18 | 2015-04-29 | Probiodrug AG | Thiourea derivatives as glutaminyl cyclase inhibitors |
US8372849B2 (en) | 2008-04-21 | 2013-02-12 | Taigen Biotechnology Co., Ltd. | Heterocyclic compounds |
WO2009146539A1 (en) * | 2008-06-02 | 2009-12-10 | Neuromed Pharmaceuticals Ltd. | 4-(aminomethyl)cyclohexanamine derivatives as calcium channel blockers |
US9023834B2 (en) | 2008-11-13 | 2015-05-05 | Taigen Biotechnology Co., Ltd. | Lyophilization formulation |
WO2011029920A1 (en) | 2009-09-11 | 2011-03-17 | Probiodrug Ag | Heterocylcic derivatives as inhibitors of glutaminyl cyclase |
WO2011107530A2 (en) | 2010-03-03 | 2011-09-09 | Probiodrug Ag | Novel inhibitors |
WO2011110613A1 (en) | 2010-03-10 | 2011-09-15 | Probiodrug Ag | Heterocyclic inhibitors of glutaminyl cyclase (qc, ec 2.3.2.5) |
WO2011131748A2 (en) | 2010-04-21 | 2011-10-27 | Probiodrug Ag | Novel inhibitors |
WO2012123563A1 (en) | 2011-03-16 | 2012-09-20 | Probiodrug Ag | Benz imidazole derivatives as inhibitors of glutaminyl cyclase |
EP3461819A1 (en) | 2017-09-29 | 2019-04-03 | Probiodrug AG | Inhibitors of glutaminyl cyclase |
US11427540B2 (en) | 2019-07-11 | 2022-08-30 | Praxis Precision Medicines, Inc. | Formulations of T-type calcium channel modulators and methods of use thereof |
US11649207B2 (en) | 2019-07-11 | 2023-05-16 | Praxis Precision Medicines, Inc. | Formulations of T-type calcium channel modulators and methods of use thereof |
US12077502B2 (en) | 2022-07-22 | 2024-09-03 | Praxis Precision Medicines, Inc. | Formulations of T-type calcium channel modulators and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
EP1007073A4 (en) | 2002-03-27 |
AU3295297A (en) | 1998-01-05 |
EP1007073A1 (en) | 2000-06-14 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6818445B2 (en) | Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) | |
US6645774B1 (en) | Methods of modifying feeding behavior using compounds with afinity for the human hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) | |
EP1007073A1 (en) | Methods of modifying feeding behavior, compounds useful in such methods, and dna encoding a hypothalamic atypical neuropeptide y/peptide yy receptor (y5) | |
US6420532B1 (en) | Method of obtaining compositions comprising Y2 specific compounds | |
US6913892B1 (en) | Method of obtaining compositions comprising Y4 specific compounds | |
WO1993010228A1 (en) | Dna encoding a glycine transporter and uses thereof | |
US5766848A (en) | Methods for identifying compounds which specifically bind a human betaine/GABA transporter | |
FI111337B (en) | DNA encoding a human serotonin receptor (5-HT4B) and its use | |
JP3501775B2 (en) | Human vanilloid receptor-like protein | |
US6713265B1 (en) | Methods of modifying feeding behavior, compounds useful in such methods, and DNA encoding a hypothalamic atypical neuropeptide Y/peptide YY receptor (Y5) |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG US UZ VN YU AM AZ BY KG KZ MD RU TJ TM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1997928786 Country of ref document: EP |
|
NENP | Non-entry into the national phase |
Ref country code: JP Ref document number: 98500766 Format of ref document f/p: F |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09194895 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: CA |
|
WWP | Wipo information: published in national office |
Ref document number: 1997928786 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1997928786 Country of ref document: EP |