CA2204491A1 - Modified neuropeptide y receptors - Google Patents
Modified neuropeptide y receptorsInfo
- Publication number
- CA2204491A1 CA2204491A1 CA 2204491 CA2204491A CA2204491A1 CA 2204491 A1 CA2204491 A1 CA 2204491A1 CA 2204491 CA2204491 CA 2204491 CA 2204491 A CA2204491 A CA 2204491A CA 2204491 A1 CA2204491 A1 CA 2204491A1
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- Prior art keywords
- receptor
- modified
- neuropeptide
- receptors
- dna
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Abstract
Modified neuropeptide Y receptors having deletions, replacements or additions in the third intracellular domain are identified and methods of making the modified receptors are provided. The invention includes the modified receptors, assays employing the modified receptors, cells expressing the modified receptors, compounds identified through the use of the modified receptors, including modulators of the receptors, and the use of the compounds to treat conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases.
Description
-CA 02204491 1997-0~-0~
WO 96/14331 PCI~/US95/14377 TITLE OF THE INVENTION
MODIFIED NEUROP~l ll)E Y RECEPTORS
BACKGROUND OF THE INVENTION
S This application is a contiml~ion-in-part of U.S. Serial No.
08/335,017, filed November 7, 1994, the contents of which are hereby incorporated by reference.
Neuropeptide Y (NPY) is a 36 residue, amidated peptide.
It is anatomically co-distributed and co-released with norepinephrine in 10 and from sympathetic postganglionic neurons ([1], [2], [3], [4], [5], [6]).
Stimulation of the sympathetic nervous system under physiological circumstances such as exercise ([7], [8]) or exposure to the cold ([9], [10]) promotes an elevation of both norepinephrine and NPY.
NPY is believed to act in the regulation of appetite control 15 ([11], [12]) and vascular smooth muscle tone ([13], [14]) as well as regulation of blood pressure ([6], [15], [16], [17]). NPY also decreases cardiac contractility ([18], [19], [20], [21], [22]). Congestive heart failure and cardiogenic shock are associated with probable releases of NPY into the blood ([23], [24], [25]). Regulation of NPY levels may be 20 beneficial to these disease states [26].
At the cellular level, neuropeptide Y binds to a G-protein coupled receptor ([27], [28], [29], [30]). Neuropeptide Y is involved in reg~ tin.~ eating behavior and is an extremely potent orixigenic agent ([11], [12], [31]). When 2-1rnini~tered intracerebroventricularly or 25 injected into the hypoth~l~mic paraventricular nucleus (PVN) it elicits eating in satiated rats ([32], [33], [34]) and intraventricular injection of antisera to NPY decreases eating ([11], [31]). It has been shown to stimulate appetite in a variety of species and at different stages of development ([12]). Other effects on energy metabolism include 30 decreased thermogenesis, body temperature and uncoupling protein, and increased white fat storage and lipoprotein lipase activity ([9], [35], [36], [37], [38], [39]). NPY levels in the PVN increase upon fasting ([40], [41], [42], [43], [44]), before a scheduled meal ([31], [36], [40]), and in both streptozotocin-induced and spontaneous diabetes ([36], [45], [46], 35 [47], [48], [49]). Also, NPY levels are increased in genetically obese CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95/14377 and hyperphagic Zucker rats ([36], [50], [51]). Thus, a specific centrally acting antagonist for the ~l~rop~iate NPY receptor subtype may be therapeutically useful for treating obesity and diabetes. Other disorders which might be targeted therapeutically include anxiety, 5 hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases ([26], [52]).
At least four receptor subtypes of the NPY family have 10 been proposed based on ph~ cological and physiological properties.
The Y1 receptor is stimulated by NPY or PYY (peptide YY) and appears to be the major vascular receptor ([16], [53], [54], [55]). The Y2 receptor is stimulated by C-terminal fragments of NPY or PYY and is abundantly expressed both centrally and peripherally ([55], [56], [57], 15 [58]). A third receptor (Y3) is exclusively responsive to NPY and is likely present in adrenal medulla, heart, and brain stem ([27], [59]). In addition, other subtypes of this receptor family are known to exist, based on pharmacological and physiological characterization ([60], [61], [62], [63]). The feeding behavior is stimulated potently by NPY, NPY2-20 36 and the Y1 agonist [Leu31, Pro34]NPY, but is not stimulated by theY2 agonist NPY13-36 ([11], [64], [65], [66]). This pharmacology is not characteristic of the defined Y1, Y2 or Y3 receptors and can thus be attributed to a unique receptor, termed "atypical Y1 " ([11], [65], [66]), that is responsible for evoking the feeding response. In addition, data 25 indicate the existence of additional members of this receptor family including one subtype specific for peptide PP ([62], [63]), one with affinity for short C-terminal fragments of NPY which induce hypotension when ~dmini~tered systemically ([15], [17], [30], [67], [68]), and one associated with binding of NPY and PYY to brain sigma and 30 phencyclidine binding sites ([61]).
The DNA encoding the Y 1 receptor has been cloned and shown to be a G protein coupled receptor ([53], [69], [70]). G-protein coupled receptors are well-known to share substantial sequence homology to each other (71). Recently, DNA encoding the Y4 receptor WO g6/14331 PCI~/US95/14377 has been isolated using Yl DNA probes [72]. In addition, DNA
encoding the Y2 receptor has been isolated by expression cloning ([73], ~74]). The cDNAs encoding these receptors are at least 45% identical at ~e DNA level and 30% at the protein level. Other NPY receptors 5 have not been cloned.
References 1. DeQuidt, M.E. and P.C. Emson, Distribution of neuropeptide Y-10 like immunoreactivity in the rat central nervous system~Immunohistochemical analysis. Neuroscience, 1986. 18(3): p. 545-618.
WO 96/14331 PCI~/US95/14377 TITLE OF THE INVENTION
MODIFIED NEUROP~l ll)E Y RECEPTORS
BACKGROUND OF THE INVENTION
S This application is a contiml~ion-in-part of U.S. Serial No.
08/335,017, filed November 7, 1994, the contents of which are hereby incorporated by reference.
Neuropeptide Y (NPY) is a 36 residue, amidated peptide.
It is anatomically co-distributed and co-released with norepinephrine in 10 and from sympathetic postganglionic neurons ([1], [2], [3], [4], [5], [6]).
Stimulation of the sympathetic nervous system under physiological circumstances such as exercise ([7], [8]) or exposure to the cold ([9], [10]) promotes an elevation of both norepinephrine and NPY.
NPY is believed to act in the regulation of appetite control 15 ([11], [12]) and vascular smooth muscle tone ([13], [14]) as well as regulation of blood pressure ([6], [15], [16], [17]). NPY also decreases cardiac contractility ([18], [19], [20], [21], [22]). Congestive heart failure and cardiogenic shock are associated with probable releases of NPY into the blood ([23], [24], [25]). Regulation of NPY levels may be 20 beneficial to these disease states [26].
At the cellular level, neuropeptide Y binds to a G-protein coupled receptor ([27], [28], [29], [30]). Neuropeptide Y is involved in reg~ tin.~ eating behavior and is an extremely potent orixigenic agent ([11], [12], [31]). When 2-1rnini~tered intracerebroventricularly or 25 injected into the hypoth~l~mic paraventricular nucleus (PVN) it elicits eating in satiated rats ([32], [33], [34]) and intraventricular injection of antisera to NPY decreases eating ([11], [31]). It has been shown to stimulate appetite in a variety of species and at different stages of development ([12]). Other effects on energy metabolism include 30 decreased thermogenesis, body temperature and uncoupling protein, and increased white fat storage and lipoprotein lipase activity ([9], [35], [36], [37], [38], [39]). NPY levels in the PVN increase upon fasting ([40], [41], [42], [43], [44]), before a scheduled meal ([31], [36], [40]), and in both streptozotocin-induced and spontaneous diabetes ([36], [45], [46], 35 [47], [48], [49]). Also, NPY levels are increased in genetically obese CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95/14377 and hyperphagic Zucker rats ([36], [50], [51]). Thus, a specific centrally acting antagonist for the ~l~rop~iate NPY receptor subtype may be therapeutically useful for treating obesity and diabetes. Other disorders which might be targeted therapeutically include anxiety, 5 hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases ([26], [52]).
At least four receptor subtypes of the NPY family have 10 been proposed based on ph~ cological and physiological properties.
The Y1 receptor is stimulated by NPY or PYY (peptide YY) and appears to be the major vascular receptor ([16], [53], [54], [55]). The Y2 receptor is stimulated by C-terminal fragments of NPY or PYY and is abundantly expressed both centrally and peripherally ([55], [56], [57], 15 [58]). A third receptor (Y3) is exclusively responsive to NPY and is likely present in adrenal medulla, heart, and brain stem ([27], [59]). In addition, other subtypes of this receptor family are known to exist, based on pharmacological and physiological characterization ([60], [61], [62], [63]). The feeding behavior is stimulated potently by NPY, NPY2-20 36 and the Y1 agonist [Leu31, Pro34]NPY, but is not stimulated by theY2 agonist NPY13-36 ([11], [64], [65], [66]). This pharmacology is not characteristic of the defined Y1, Y2 or Y3 receptors and can thus be attributed to a unique receptor, termed "atypical Y1 " ([11], [65], [66]), that is responsible for evoking the feeding response. In addition, data 25 indicate the existence of additional members of this receptor family including one subtype specific for peptide PP ([62], [63]), one with affinity for short C-terminal fragments of NPY which induce hypotension when ~dmini~tered systemically ([15], [17], [30], [67], [68]), and one associated with binding of NPY and PYY to brain sigma and 30 phencyclidine binding sites ([61]).
The DNA encoding the Y 1 receptor has been cloned and shown to be a G protein coupled receptor ([53], [69], [70]). G-protein coupled receptors are well-known to share substantial sequence homology to each other (71). Recently, DNA encoding the Y4 receptor WO g6/14331 PCI~/US95/14377 has been isolated using Yl DNA probes [72]. In addition, DNA
encoding the Y2 receptor has been isolated by expression cloning ([73], ~74]). The cDNAs encoding these receptors are at least 45% identical at ~e DNA level and 30% at the protein level. Other NPY receptors 5 have not been cloned.
References 1. DeQuidt, M.E. and P.C. Emson, Distribution of neuropeptide Y-10 like immunoreactivity in the rat central nervous system~Immunohistochemical analysis. Neuroscience, 1986. 18(3): p. 545-618.
2. Lundberg, J.M., et al., Co-release of neuropeptide Y and catecholamines during physical exercise in man. Biochem Biophys Res 15 Commun, 1985. 133(1): p. 30-6.
3. Morris, M.J., et al., Increases in plasma neuropeptide Y
concentrations during sympathetic activation in man. J Auton Nerv Syst, 1986. 17(2): p. 143-9.
concentrations during sympathetic activation in man. J Auton Nerv Syst, 1986. 17(2): p. 143-9.
4. Pemow, J., Co-release andfunctional interactions of neuropeptide Y and noradrenaline in peripheral sympathetic vascular control. Acta Physiol Scand Suppl, 1988. 568(1): p. 1-56.
25 5. Sawchenko, P.E., et al., Colocalization of neuropeptide Y
immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricl~lar nucleus of the hypothalamus. J Comp Neurol, 1985. 241(2): p. 138-53.
30 6. Wahlestedt, C., et al., Norepinephrine and neuropeptide Y:
vasoconstrictor cooperation in vivo and in vitro. Am J Physiol, 1990.
258: p. R736-R742.
7. Kaijser, L., et al., Neuropeptide Y is released together with 35 noradrenaline from the human heart during exercise and hypoxia. Clin Physiol, 1990. 10(2): p. 179-88.
8. Lewis, D.E., et al., Intense exercise and food restriction cause similar hypothalamic neuropeptide Y increases in rats. Am J Physiol, 40 1993. 264: p. E279-E284.
CA 02204491 1997-0~-0~
9. McCarthy, H.D., et al., Widespread increases in regional hypothalamic Neuropeptide- Y levels in acute Cold-Exposed rats.
Neuroscience, 1993. 54(1): p. 127-132.
10. Zukowska, G.Z. and A.C. Vaz, Role of neuropeptide Y (NPY) in cardiovascular responses to stress. Synapse, 1988. 2(3): p. 293-8.
11. Stanley, B.G., et al., Evidence for neuropeptide Y mediation of 0 eating produced by food deprivation and for a variant of the Yl receptor mediating this peptide's effect. Peptides, 1992. 13: p. 581-587.
12. Stanley, B.G., Neuropeptide Y in multiple hypothalamic sites controls eating behavior, endocrine, and autonomic systems for body 15 energy balance, in Neuropeptide Y, W.F. Colmers and C. Wahlestedt, Editor. 1993, Hllm~n~ Press: Totowa, NJ. p. 457-509.
13. Abel, P.W. and C. Ha~, Effects of neuropeptide Y on contraction, relaxation, and membrane potential of rabbit cerebral arteries. J
20 Cardiovasc Pharmacol, 1989. 13(1): p. 52-63.
14. Han, C. and P.W. Abel, Neuropeptide Y potentiates contraction and inhibits relaxation of rabbit coronary arteries. J Cardiovasc Pharmacol, 1987. 9(6): p. 675-81.
15. Grundemar, L., et al., Biphasic blood pressure response to neuropeptide Y in anesthetized rats. Eur J Pharmacol, 1990. 179(1-2):
p. 83-7.
30 16. Grundemar, L., et al., Characterization of vascular neuropeptide Y receptors. Br J Pharmacol, 1992. 105(1): p. 45-50.
17. Shen, S.H., et al., C-terminal neuropeptide Y fragments are mast cell-dependent vasodepressor agents. Eur. J. Pharmacol., 1993. 204: p.
35 249-256.
18. Tseng, C.J., et al., Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ Res, 1989. 64(1): p. 55-61.
40 19. Carter, D.A., M. Vallejo, and S.L. Lightman, Cardiovascular effects of neuropeptide Y in the nucleus tractus solitarius of rats:
CA 02204491 1997~ -0~
relationship with noradrenaline and vasopressin. Peptides, 1985. 6(3):
p. 421-5.
20. Grundemar, L., C. Wahlestedt, -nd D.J. Reis, Neuropeptide Y
25 5. Sawchenko, P.E., et al., Colocalization of neuropeptide Y
immunoreactivity in brainstem catecholaminergic neurons that project to the paraventricl~lar nucleus of the hypothalamus. J Comp Neurol, 1985. 241(2): p. 138-53.
30 6. Wahlestedt, C., et al., Norepinephrine and neuropeptide Y:
vasoconstrictor cooperation in vivo and in vitro. Am J Physiol, 1990.
258: p. R736-R742.
7. Kaijser, L., et al., Neuropeptide Y is released together with 35 noradrenaline from the human heart during exercise and hypoxia. Clin Physiol, 1990. 10(2): p. 179-88.
8. Lewis, D.E., et al., Intense exercise and food restriction cause similar hypothalamic neuropeptide Y increases in rats. Am J Physiol, 40 1993. 264: p. E279-E284.
CA 02204491 1997-0~-0~
9. McCarthy, H.D., et al., Widespread increases in regional hypothalamic Neuropeptide- Y levels in acute Cold-Exposed rats.
Neuroscience, 1993. 54(1): p. 127-132.
10. Zukowska, G.Z. and A.C. Vaz, Role of neuropeptide Y (NPY) in cardiovascular responses to stress. Synapse, 1988. 2(3): p. 293-8.
11. Stanley, B.G., et al., Evidence for neuropeptide Y mediation of 0 eating produced by food deprivation and for a variant of the Yl receptor mediating this peptide's effect. Peptides, 1992. 13: p. 581-587.
12. Stanley, B.G., Neuropeptide Y in multiple hypothalamic sites controls eating behavior, endocrine, and autonomic systems for body 15 energy balance, in Neuropeptide Y, W.F. Colmers and C. Wahlestedt, Editor. 1993, Hllm~n~ Press: Totowa, NJ. p. 457-509.
13. Abel, P.W. and C. Ha~, Effects of neuropeptide Y on contraction, relaxation, and membrane potential of rabbit cerebral arteries. J
20 Cardiovasc Pharmacol, 1989. 13(1): p. 52-63.
14. Han, C. and P.W. Abel, Neuropeptide Y potentiates contraction and inhibits relaxation of rabbit coronary arteries. J Cardiovasc Pharmacol, 1987. 9(6): p. 675-81.
15. Grundemar, L., et al., Biphasic blood pressure response to neuropeptide Y in anesthetized rats. Eur J Pharmacol, 1990. 179(1-2):
p. 83-7.
30 16. Grundemar, L., et al., Characterization of vascular neuropeptide Y receptors. Br J Pharmacol, 1992. 105(1): p. 45-50.
17. Shen, S.H., et al., C-terminal neuropeptide Y fragments are mast cell-dependent vasodepressor agents. Eur. J. Pharmacol., 1993. 204: p.
35 249-256.
18. Tseng, C.J., et al., Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ Res, 1989. 64(1): p. 55-61.
40 19. Carter, D.A., M. Vallejo, and S.L. Lightman, Cardiovascular effects of neuropeptide Y in the nucleus tractus solitarius of rats:
CA 02204491 1997~ -0~
relationship with noradrenaline and vasopressin. Peptides, 1985. 6(3):
p. 421-5.
20. Grundemar, L., C. Wahlestedt, -nd D.J. Reis, Neuropeptide Y
5 acts at an atypical receptor to evoke cardiovascular depression and to inhibit glutamate responsiveness in the brainstem. J Ph~ col Exp Ther, 1991. 258(2): p. 633-8.
21. Grundemar, L., C. Wahlestedt, a nd D.J. Reis, Long-lasting 0 inhibition of the cardiovascular responses to glutamate and the baroreceptor reflex elicited by neurop~ptide Y injected into the nucleus tractus solitarius of the rat. Neurosci Lett, 1991. 122(1): p. 135-9.
22. Zukowska-Grojec, Z. and C. Wallestedt, Origin and actions of 15 neuropeptide Y in the cardiovascular ~ystem, in Neuropeptide Y, W.F.
Colmers and C. Wahlestedt, Editor. 1993, Hllm~n~ Press: Totowa, NJ.
p. 315-388.
23. Edvinsson, L., et al., Congestive heart failure: involvement of 20 perivascular peptides reflecting activi~ in sympathetic, parasympathetic and afferentfibres. Eur J Clin Invest, 990. 20(1): p. 85-9.
24. Franco, C.A., et al., Release of nPuropeptide Y and noradrenaline from the human heart after aortic occlusion during coronary artery 25 surgery. Cardiovasc Res, 1990. 24(3): ?. 242-6.
25. Maisel, A.S., et al., Elevation of ~lasma neuropeptide Y levels in congestive heartfailure. Am J Med, lC89. 86(1): p. 43-8.
30 26. Wahlestedt, C. and D.J. Reis, Neuropeptide Y-related peptides and their receptors - are the receptors potential therapeutic drug targets? Annu. Rev. Ph~ col. Toxicol., 1993. 32: p. 309-352.
27. Wahlestedt, C., S. Regun~th~n, and D.J. Reis, Identification of 35 cultured cells selectively expressing Y1-, Y2-, or Y3-type receptors for neuropeptide Y/peptide YY. Life Sciences, 1992. 50: p. PL7-PL12.
28. Feth, F., W. Rascher, and M.C. Michel, G-protein coupling and signalling of Y1-like neuropeptide Y receptors in SK-N-MC cells.
40 Naunyn Schmiedebergs Arch Pharmaccl, 1991. 344(1): p. 1-7.
CA 02204491 1997~ -0~
29. Motulsky, H.J. and M.C. Michel. Neuropeptide Y mobilizes Ca2+
and inhibits adenylate cyclase in human erythroleukemia cells. Am J
Physiol, 1988. 255: p. E880-E885.
5 30. Wahlestedt, C., et al., Neuropeptide Y receptor subtypes, Yl and Y2. Ann N Y Acad Sci, 1990. 611(7): p. 7-26.
31. Sahu, A. and S.P. Kalra, Neurop~ptidergic regulation of feeding-behavior - neuropeptide-Y. Trends In Endocrinology And Metabolism, 10 1993. 4(7): p. 217-224.
32. Clark, J.T., et al., Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology, 1984.
115(1): p. 427-429.
33. Stanley, B.G. and S.F. Leibowitz, Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant offeeding behavior. Proc. Natl. Acad. Sci. USA, 1985. 82: p. 3940-3943.
20 34. Stanley, B.G. and S.F. Leibowitz, Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci, 1984. 35(26): p. 2635-42.
35. Zarjevski, N., et al., Chronic intracerebroventricular 25 neuropeptide-Y administration to normal rats mimics hormonal and metabolic changes of obesity. Endocrinology, 1993. 133(4): p. 1753-1758.
i 36. Billington, C.J. and A.S. Levine, Hypothalamic neuropeptide Y
30 regulation offeeding and energy metabolism. Current Opinion in Neurobiology, 1992. 2: p. 847-851.
37. Leibowitz, S.F., Brain neuropeptide Y: an integrator of endocrine, metabolic and behavioral processes. Brain Research Bulletin, 35 1991. 27: p. 333-337.
38. Billington, C.J., et al., Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am. J. Physiol., 1991. 260:
p. R321-R327.
CA 02204491 1997-0~-0~
WO 96/14331 PCI'/US95/14377 39. Billington, C.J., et al., Neuropeptide-Y in hypothalamic paraventricular nucleus - a center coordinating energy-metabolism.
American Journal Of Physiology, 1994. 266(6): p. R1765-R1770.
5 40. Kalra, S.P., et al., Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food.
Proc. Natl. Acad. Sci. USA, 1991. 88: p. 10931-10935.
41. Beck, B., et al., Rapid and localized alterations of neuropeptide Y
0 in discrete hypothalamic nuclei with feeding status. Brain Res, 1990.
528(2): p. 245-9.
42. Brady, L.S., et al., Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats.
15 Neuroendocrinology, 1990. 52(5): p. 441-7.
43. Calza, L., et al., Increase of neuropeptide Y-like immunoreactivity in the paraventricular nucleus offasting rats.
Neurosci Lett, 1989. 104(1-2): p. 99-104.
44. Sahu, A., P.S. Kalra, and S.P. Kalra, Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus. Peptides, 1988. 9(1): p. 83-6.
25 45. Abe, M., et al., Increased neuropeptide Y content in the arcuato-paraventricular hypothalamic neuronal system in both insulin-dependent and non-insulin-dependent diabetic rats. Brain Res, 1991. 539(2): p.
223-7.
30 46. Sahu, A., et al., Neuropeptide-Y concentration in microdissected hypothalamic regions and in vitro release from the medial basal hypothalamus-preoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy. Endocrinology, 1990. 126(1): p.
192-8.
47. White, J.D., et al., Increased hypothalamic content of preproneuropeptide-Y messenger ribonucleic acid in streptozotocin-diabetic rats. Endocrinology, 1990. 126(2): p. 765-72.
40 48. Willi~m.~, G., et al., Increased hypothalamic neuropeptide Y
concentrations in diabetic rat. Diabetes, 1988. 37(6): p. 763-72.
CA 02204491 1997-o~-o~
49. Willi~m.~, G., et al., Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats.
Diabetes, 1989. 38(3): p. 321-7.
50. Beck, B., et al., Hypothalamic neuropeptide Y (NPY) in obese Zucker rats: implications in feeding and sexual behaviors. Physiol Behav, 1990. 47(3): p. 449-53.
10 51. Sanacora, G., et al., Increased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation. Endocrinology, 1990. 127(2): p. 730-7.
15 52. Wahlestedt, C., R. F.km~n, and E. Widerlov, Neuropeptide Y
(NPY) and the central nervous system: distribution effects and possible relationship to neurological and psychiatric disorders. Prog Neuropsychoph~ col Biol Psychiatry, 1989. 13(1-2): p. 31-54.
20 53. Larh~mm~r, D., et al., Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Yl-type. J. Biol.
Chem., 1992. 267: p. 10935- 10938.
54. Sheikh, S.P., et al., Localization of Y] receptors for NPY and 25 Pn on vascular smooth muscle cells in rat pancreas. Arn J Physiol, 1991. 260: p. G250-G257.
55. Wahlestedt, C., N. Y~n~ih~ra, and R. Hakanson, Evidence for diJ~ferent pre-and post-junctional receptors for neuropeptide Y and 30 related peptides. Regul Pept, 1986. 13(3-4): p. 307-18.
56. Jorgensen, J.C., J. Fuhlendorff, and T.W. Schwartz, Structure-function studies on neuropeptide Y and pancreatic polypeptide--evidence for two PP-fold receptors in vas deferens. Eur J Ph~ Gol, 1990.
35 186(1): p. 105-14.
57. Cox, H.M. and J.L. Krstenansky, The effects of selective amino acid substitution upon neuropeptide Y antisecretory potency in rat jejunum mucosa. Peptides, 1991. 12(2): p. 323-7.
CA 02204491 1997-0~-0~
58. Aicher, S.A., et al., Receptor-selective analogs demonstrate NPYIPYY receptor heterogeneity in rat brain. Neurosci Lett, 1991.
130(1): p. 32-6.
5 59. Balasubr2m~ni~m, A., et al., Characterization of neuropeptide Y
binding sites in rat cardiac ventricular membranes. Peptides, l 990.
11(3): p. 545-50.
60. Li, X.J., et al., Cloning, functional expression, and developmental 0 regulation of a neuropeptide Y receptor from Drosophila melanogaster.
J Biol Chem, 1992. 267(1): p. 9-12.
61. Roman, F.J., et al., Neuropeptide Y and peptide YY interact with rat brain sigma and PCP binding sites. Eur J Pharmacol, 1989. 174(2-15 3): p. 301-2.
62. Schwartz, T.W., S.P. Sheikh, and M.M. O'Hare, Receptors on phaeochromocytoma cells for two members of the PP-fold family--NPY
and PP. Febs Lett, 1987. 225(1-2): p. 209-14.
63. Schwartz, T.W., et al., Signal epitopes in the three-dimensional structure of neuropeptide Y. Interaction with Yl, Y2, and pancreatic polypeptide receptors. Ann N Y Acad Sci, 1990. 611(35): p. 35-47.
25 64. Wahlestedt, C., et al., Modulation of anxiety and neuropeptide Y-Yl receptors by antisense oligodeoxynucleotides. Science, 1993. 259: p.
528-531.
65. Jolicoeur, F.B., et al., In vivo structure activity study supports the 30 existence of heterogeneous neuropeptide Y receptors. Brain Res Bull, 1991. 26(2): p. 309-11.
66. Leibowitz, S.F. and J.T. Alexander, Analysis of neuropeptide Y-inducedfeeding: dissociation of Yl and Y2 receptor effects on natural 35 meal patterns. Peptides, 1991. 12(6): p. 1251 -60.
67. Inui, A., et al., Characterization of peptide YY receptors in the brain. Endocrinology, 1989. 124(1): p. 402-9.
CA 02204491 1997-0~-0~
WO 96/14331 PCI~/US95/14377 68. Boublik, J., et al., Neuropeptide Y and ne~lropeptide Yl~-36.
Structural and biological characterization. Int J Pept Protein Res, 1989.
33(1): p. 11-5.
5 69. Eva, C., et al., Molecular cloning of a novel G protein-coupled receptor that may belong to the neuropeptide receptor family. FEBS
Lett., 1990. 271: p. 81-84.
70. Herzog, H., et al., Cloned human neuropeptide Y receptor couples 1O to two di~erent second messenger systems. Proc Natl Acad Sci U S A, 1992. 89: p. 5794-5798.
71. Strader, C.D., I.S. Sigal, and R.A. Dixon, Structural basis of beta-adrenergic receptor function. FASEB-J, 1989. 3(7): p. 1825-1832.
72. Bard, J.A., Walker, M.W., Brancheck, T., Wein~h~nk, R. DNA
encoding a human neuropeptide Ylpeptide YYIpancreatic polypeptide receptor (Y4) and uses thereof. PCT International Application Publication No. WO 95/17906, published 6 July 1995.
73. Gerald, C., Walker, M.W., Branchek, T., and Weinshank, R.
Nucleic acid encoding Neuropeptide YlPeptide YY (Y2( receptors and uses thereof. PCT International Application Publication No.
WO95/21245, published 10 August 1995.
74. Rose, P. A. et al., Cloning and functional expression of a cDNA
encoding the human type 2 neuropeptide Y receptor. J. Biol. Chem., 270:22661 -22664.
Modified neuropeptide Y receptors having deletions, replacements or additions in the third intracellular domain are identified and methods of making the modified receptors are provided. The invention includes the modified receptors, assays employing the 35 modified receptors, cells expressing the modified receptors, compounds identified through the use of the modif1ed receptors, including modulators of the receptors, and the use of the compounds to treat conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95114377 cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases.
BREF DESCRIPTION OF THE DRAWINGS
5 Figure 1. Schematic diagram of G-protein signal transduction system.
The receptor is shown as a seven-helical bundle. a, ~, and ~ indicate the three subunits of the G protein. E indicates an effector enzyme, such as adenylyl cyclase. The agonist ~A) binding with high affinity to the receptor-G protein complex and with low affinity to the receptor 10 alone is shown.
Figure 2. Schematic diagram of the hamster ~2 adrenergic receptor.
The third intracellular loop comprises residues 221-273. The proximal and distal segments of this loop are drawn in cylinders.
Figure 3 shows the amino acid sequence of the human NPY1 receptor subtype aligned with that of the hamster ~2-adrenergic receptor. The transmembrane helices are underlined.
Modified neuropeptide Y receptors having deletions, replacements or additions in the third intracellular domain are identified and methods of m~king the modified receptors are provided. The invention includes the modified receptors, assays employing the 25 modified receptors, cells expressing the modified receptors, compounds identified through the use of the modified receptors, including modulators of the receptors, and the use of the compounds to treat conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and 30 cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases. Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers.
CA 02204491 1997-0~-0~
WO 96/14331 PCI/US95tl4377 Neuropeptide Y receptors belong to a class of receptors known as "G-protein coupled receptors." The term "G-protein coupled receptor" refers to any receptor protein that mediates its endogenous signal transduction through activation of one or more guanine nucleotide binding regulatory proteins (G-proteins). These receptors share common structural features, including seven hydrophobic transmembrane domains. G-protein coupled receptors include receptors that bind to small biogenic amines, including but not limited to beta-adrenergic receptors (~AR), alpha-adrenergic receptors (ocAR) and muscarinic receptors, as well as receptors whose endogenous ligands are peptides, such as neurokinin, neuropeptide Y and glucagon receptors.
Examples of ~AR include beta-1, beta-2, and beta-3 adrenergic receptors.
G-protein coupled receptors are cell surface proteins that mediate the responses of a cell to a variety of environmental signals.
Upon binding an agonist, the receptor interacts with one or more specific G proteins, which in turn regulate the activities of specific effector proteins. By this means, activation of G-protein coupled receptors amplifies the effects of the environmental signal and initiates a cascade of intracellular events that ultimately leads to defined cellular responses. G-protein coupled receptors function as a complex information processing network within the plasma membrane of the cell, acting to coordinate a cell's response to multiple environmental signals.
G-protein coupled receptors are characterized by the ability of agonists to promote the formation of a high affinity ternary complex between the agonist, the receptor and the G-protein (Figure 1). The a-subunit of the G protein contains a guanine nucleotide binding site which, in the high affinity ternary [G protein-receptor-agonist]
complex, is occupied by GDP. In the presence of physiological concentrations of GTP, the GDP molecule in the guanine nucleotide binding site of the G protein is displaced by a GTP molecule. The binding of GTP dissociates the a subunit of the G protein from its ~ and y subunits and from the receptor, thereby activating the G-protein to CA 02204491 1997-0~-0~
stimulate downstream effectors (adenylyl cyclase in the case of the ,B-adrenergic receptor (~AR)) and propagating the intracellular signal.
Thus, the ternary complex is transient in the presence of physiological concentrations of GTP. Because the affinity of the agonist for the 5 receptor-G protein complex is higher than its affinity for the uncomplexed receptor, one consequence of the destabilization of the ternary complex is a reduction in the affinity of the receptor for the agonist. Thus, the affinity of agonists for G-protein coupled receptors is a function of the efficiency with which the receptor is coupled to the 10 G-protein. In contrast, antagonists bind with the same affinity to the receptor in the presence or absence of G-protein coupling.
The observation that agonist affinity can be reduced by conditions under which a receptor is not optimally coupled to its G-protein has important implications for the identification of agonists of 15 G-protein coupled receptors, particularly identification based on ligand binding. If a receptor is not optimally coupled to the G-protein under the conditions of binding assays, an agonist will bind to the receptor with relatively low affinity. Thus, a screen that relies on a binding assay based on displacement of a radiolabeled ligand, although attractive 20 for its ease and the potential for high throughput, poses the risk that a promising partial agonist might be overlooked because the agonist would bind predominantly to the low affinity state of the receptor, and thus would have low affinity in the binding assay. Consequently, functional assays are frequently used to screen for agonists of G-protein 25 coupled receptors. However, functional assays (ranging from ex vivo muscle contraction assays to determination of second messenger levels in cells expressing exogenous cloned G-protein coupled receptors) are tedious and more time-consuming than ligand binding assays, and hence are not readily adapted to high-throughput screens. Because the 30 modified receptors of the present invention bind agonists with high affinity in the presence or absence of the G-protein, they can be used in high throughput radioligand binding assays to screen for high affinity ligands, regardless of whether the ligands are agonists or antagonists.
CA 02204491 1997-0~-0~
G-protein coupled receptors consist of seven hydrophobic domains connecting eight hydrophilic domains. The hydrophobicity or hydrophilicity of the domains may be determined by standard hydropathy profiles, such as Kyte-Doolittle analysis (Kyte, J. and 5 Doolittle, R.J.F. J. Mol. Biol. 157: 105 (1982)). The receptors are thought to be oriented in the plasma membrane of the cell such that the N-terminus of the receptor faces the extracellular space and the C-terminus of the receptor faces the cytoplasm, so that each of the hydrophobic domains crosses the plasma membrane. The receptors 10 have been modeled and the putative boundaries of the extracellular, transmembrane and intracellular domains are generally agreed (for a review, see Baldwin, EMBO J. 12:1693, 1993). In general, the transmembrane domains are comprised of stretches of 20-25 amino acids in which most of the amino acid residues have hydrophobic side 15 chains (including cysteine, methionine, phenyl~l~nine, tyrosine, tryptophan, proline, glycine, ~l~nine, valine, leucine, isoleucine), whereas the intracellular and extracellular loops are defined by contiguous stretches of several amino acids that have hydrophilic or polar side chains (including aspartate, glutamate, asparagine, glutamine, 20 serine, threonine, histidine, lysine, and arginine). Polar amino acids, especially uncharged ones (such as serine, threonine, asparagine, and gll-t~mine) are found in both transmembrane and extramembrane regions.
The extramembrane regions are characterized by 25 contiguous stretches of three or more hydrophilic residues. In contrast, hydrophilic residues are found only in groups of 1-2, surrounded by hydrophobic residues, in the transmembrane domain. Thus, the transmembrane and extramembrane regions can be identified by the number of contiguous hydrophilic or hydrophobic amino acids in the 30 primary sequence of the receptor, in addition to the constraints on the length of the hydrophobic segments given above. The boundaries between the transmembrane and extramembrane regions are often defined by the presence of charged or polar residues at the beginning or end of a stretch of hydrophobic amino acids. The locations of the CA 02204491 1997-0~-0~
mutations in the receptors of the present invention are described on the basis of these models and can be specifically defined by the specific amino acid numbers of the residues being mllt~ted.
By these criteria, the third intracellular loop is defined as 5 the hydrophilic loop connecting the hydrophobic, putative tr~n~membrane domains V and VI. For example, in hamster ,~2 adrenergic receptor, the third intracellular loop would refer to amino acids 221 through 273. In accordance with the principles described above, the beginning of this loop is defined by the presence of Arg22 10 (a charged residue at the end of the hydrophobic stretch of residues 198-220) and Lys273 (a charged residue at the beginning of the hydrophobic stretch of residues 274-298). In the human NPY1 receptor (PCT International Application Publication Nos. W093/09227 published 13 May 1993 and WO93/24515 published 9 December 1993, the 15 contents of both of which are hereby incorporated by reference), the third intracellular loop refers to amino acids #233-260 (Figure 3). In accordance with the principles described above, the beginning of this loop is defined by the presence of Lys233 (a charged residue at the end of the long stretch of hydrophobic residues comprising helix 5) and 20 Arg260 (a charged residue at the beginning of the long stretch of hydrophobic residues comprising helix 6). In the rat NPY1 receptor, the third intracellular loop refers to amino acids #232-259 (Eva, C., et al., FEBS Lett. 271:81, 1990). In accordance with the principles described above, the be~inning of this loop is defined by the presence of 25 Lys232 (a charged residue at the end of the long stretch of hydrophobic residues comprising helix 5) and Arg259 (a charged residue at the beginning of the long stretch of hydrophobic residues comprising helix 6). In the human and rat NPY2 receptors, the third intracellular loop refers to amino acids #241-268 (Gerald, C., et al., PCT International 30 Application Publication No. WO95/21245, the contents of which are hereby incorporated by reference). In accordance with the principles described above, the be~innin~ of this loop is defined by the presence of Arg241 (a charged residue at the end of the long stretch of hydrophobic residues comprising helix 5) and Lys268 (a charged residue at the CA 02204491 1997-o~-o~
WO 96/14331 PCT/US95tl4377 beginning of the long stretch of hydrophobic residues comprising helix 6). In the human NPY4 receptor, the third intracellular loop refers to amino acids #236-263 (Bard, J.A., et al., PCT International Application Publication No. WO95/17906, the contents of which are hereby 5 incorporated by reference). In accordance with the principles described above, the beginning of this loop is defined by the presence of Arg236 (a charged residue at the end of the long stretch of hydrophobic residue.s comprising helix 5) and Gln263 (a polar residue at the beginning of the long stretch of hydrophobic residues comprising helix 6). In the rat 10 NPY4 receptor, the third intracellular loop refers to amino acids #236-263 (Bard, J.A., et al., PCT International Application Publication No.
WO95/17906). In accordance with the principles described above, the beginning of this loop is defined by the presence of Arg236 (a charged residue at the end of the long stretch of hydrophobic residues 15 comprising helix 5) and Arg263 (a charged residue at the beginning of the long stretch of hydrophobic residues comprising helix 6).
The present invention pertains to modified neuropeptide Y receptors having deletions, replacements or additions in the third intracellular domain. Methods of designing and making modified 20 receptors are provided. The modified receptors are uncoupled from or are poorly coupled to their respective neuropeptides. However, these modif1ed receptors bind agonists with high affinity in the absence of G protein coupling. Because of their high intrinsic affinity for agonists, these modified receptors may be used in high 25 throughput binding assays to identify compounds that bind to the receptor with high affinity, regardless of whether these compounds are agonists or antagonists. The invention includes the DNA
encoding the modif1ed receptors, the modified receptors, assays employing the modified receptors, cells expressing the modified 30 receptors, substances identified through the use of the modified receptors including specific modulators of the modified receptors, and the use of these substances in treating diseases, including obesity, diabetes, cardiovascular, and neurological disorders. Modulators identified in this process are useful as therapeutic agents.
CA 02204491 1997-0~-05 Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers.
Modified receptors may include genetic variants, both natural and induced. Induced modified receptors may be derived by a S variety of methods, including but not limited to, site-directed mutagenesis. Techniques for nucleic acid and protein manipulation are well-known in the art and are described generally in Methods in Enzymology and in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989).
It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA
sequences which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not subst~nti~lly alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for Iysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
As used herein, a "functional derivative" of a modified receptor is a compound that possesses a biological activity (either functional or structural) that is substantially .simil~r to the biological activity of the modified receptor. The term "functional derivative"
is intended to include the "fragments," "variants," "degenerate CA 02204491 1997-0~-0~
variants," "analogs" and "homologues" or to "chemical derivatives"
of modified receptors. The term "fragment" is meant to refer to any polypeptide subset of modif1ed receptors. The term "variant" is meant to refer to a molecule subst~nti~lly similar in structure and function to either the entire modified receptor molecule or to a fragment thereof. A molecule is "subst~nti~lly similar" to a modified receptor if both molecules have subst~nti~lly similar structures or if both molecules possess similar biological activity.
Therefore, if the two molecules possess subst~nti~lly similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
The term "analog" refers to a molecule substantially similar in function to either the entire modifed receptor molecule or to a fragment thereof.
"Substantial homology" or "substantial similarity", when referring to nucleic acids means that the segments or their complementary strands, when optimally aligned and compared, are identical with a~pro~liate nucleotide insertions or deletions, in at least 50% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize to a strand or its complement.
The nucleic acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or subst~nti~lly purified form. A nucleic acid is considered substantially purified when it is purified away from environmental cont~min~nt,s. Thus, a nucleic acid sequence isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
Nucleic acid compositions of this invention may be derived from genomic DNA or cDNA, prepared by synthesis or by a combination of techniques.
The natural or synthetic nucleic acids encoding the modified G-coupled protein receptors of the present invention may be CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95/14377 incorporated into expression vectors. Usually the expression vectors incorporating the modified receptors will be suitable for replication in a host. Examples of acceptable hosts include, but are not limited to, prokaryotic and eukaryotic cells.
The phrase "recombinant expression system" as used herein means a subst~nti~lly homogenous culture of suitable host org~ni~m~ that stably carry a recombinant expression vector. Examples of suitable hosts include, but are not limited to, bacteria, yeast, fungi, insect cells, plant cells and m~mm~ n cells. Generally, cells of the 10 expression system are the progeny of a single ancestral transformed cell.
The cloned modified receptor DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector cont~inin~ a suitable 15 promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant modified receptor. Techniques for such manipulations are fully described in Sambrook, J., et al., supra, and are well known in the art.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells and 25 ~nim~l cells.
Specifically designed vectors allow the shllttling of DNA
between hosts such as bacteria-yeast or bacteria-~nim~l cells or bacteria-fungi or bacteria-invertebrate cells. An appropriately constructed expression vector should contain: an origin of replication 30 for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initi~te RNA synthesis. A strong promoter is one which causes CA 02204491 1997-0~-0~
mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
A variety of m~mm~ n expression vectors may be used to express recombinant modified receptor in m~mm~ n cells.
Commercially available m~mm~ n expression vectors which may be suitable for recombinant modified receptor expression, include but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC
37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and ~ZD35 (ATCC 37565), pCI-neo (Promega).
A variety of bacterial expression vectors may be used to express recombinant modified receptor in bacterial cells.
Commercially available bacterial expression vectors which may be suitable for recombinant modified receptor expression include, but are not limited to pET1 la (Novagen), lambda gtl 1 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to express recombinant modified receptor in fungal cells.
Commercially available fungal cell expression vectors which may be suitable for recombinant modified receptor expression include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen).
A variety of insect cell expression vectors may be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of modified receptor include but are not limited to pBlue Bac III (Invitrogen).
An expression vector cont~ining DNA encoding modified receptor may be used for expression of modified receptor in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95114377 as E. coli, fungal cells such as yeast, m~mm~ n cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from m~mm~lian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC
CCL 86), CV-l (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL
92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC
CCL 171).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-cont~ining cells are clonally propagated and individually analyzed to determine whether they produce modified receptor protein. Identification of modified receptor expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-modified receptor antibodies.
Expression of modified receptor DNA may also be performed using in vitro produced synthetic mRNA or native mRNA. Synthetic mRNA or mRNA isolated from modified receptor producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
The term "substantial homology", when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30% homology with the naturally occurring protein in question, usually at least about 40% homology.
CA 02204491 1997-0~-0~
WO 96/14331 PCI'/US95114377 The modified receptors may be expressed in an appropriate host cell and used to discover compounds that affect the modified receptor. Preferably, the modified receptors are expressed in a m~mm~ n cell line, including but not limited to, COS-7, CHO or L
5 cells, or an insect cell line, including but not limited to, Sf9 and Sf21, and may be used to discover ligands that bind to the receptor and alter or stim~ te its function. The modified receptors may also be produced in bacterial, fungal or yeast expression systems.
The expression of the modified receptor may be detected 10 by use of a radiolabeled ligand specific for the receptor. For example, for the ,~2 adrenergic receptor, such a ligand may be 125I-iodocyanopindolol (125I-CYP). For the NPY receptor, such a ligand may be 125I-NPY, 125I-Peptide YY (PYY) or 125I-Pancreatic polypeptide.
The specificity of binding of compounds showing affinity for the modified receptors is shown by measuring the affinity of the compounds for cells transfected with the cloned modified receptor or for membranes from these cells. Expression of the cloned modified receptor and screening for compounds that inhibit the binding of 20 radiolabeled ligand to these cells provides a rational way for rapid selection of compounds with high affinity for the receptor. These compounds may be agonists or antagonists of the receptor. Because the modified receptor does not couple well to G proteins, the agonist activity of these compounds is best assessed by using the wild-type 25 receptor, either natively expressed in tissues or cloned and exogenously expressed.
Once the modified receptor is cloned and expressed in a m~mm~ n cell line, such as COS-7 cells or CHO cells, the recombinant modified receptor is in a well-characterized environment. The 30 membranes from the recombinant cells expressing the modified receptor are then isolated according to methods known in the art. The isolated membranes may be used in a variety of membrane-based receptor binding assays. Because the modified receptor has a high affinity for agonists, ligands (either agonists or antagonists) may be CA 02204491 1997-0~-0~
identified by standard radioligand binding assays. These assays will measure the intrinsic affinity of the ligand for the receptor.
The present invention provides methods of generating modified NPY receptors. Such methods generally comprise the deletion 5 of at least one nucleotide from the third intracellular domain of the receptor. Additional methods include, but are not limited to, enzymatic or chemical removal of amino acids from the third intracellular domain of the receptor. One method of generating modified NPY receptors comprlses:
(a) isolating DNA encoding an NPY receptor;
(b) altering the DNA of step (a) by deleting at least one nucleotide from DNA encoding the third intracellular domain of the NPY receptor or disrupting the amphipathic helix at the N- or C-terminus of the third intracellular domain by replacement with 15 nucleotides or addition of nucleotides coding for non-helical protein sequence;
(c) isolating the altered DNA;
(d) expressing the altered DNA; and (e) recovering the modified NPY receptor.
20 The third intracellular domain of a G-protein coupled receptor is located between the fifth and sixth hydrophobic transmembrane domains of the receptor.
The present invention provides methods of identifying compounds that bind to modified NPY receptors. Methods of 25 identifying compounds are exemplified by an assay, comprising:
a) cloning a neuropeptide Y receptor;
b) altering the DNA sequence encoding the third intracellular domain of the cloned receptor;
c) splicing the altered receptor into an expression 30 vector to produce a construct such that the altered receptor is operably linked to transcription and translation signals suff cient to induce expression of the receptor upon introduction of the construct into a prokaryotic or eukaryotic cell;
CA 02204491 1997-0~-0~
WO 96/14331 PCI~/US95/14377 d) introducing the construct into a prokaryotic or eukaryotic cell which does not express the altered receptor in the absence of the introduced construct; and e) incubating cells or membranes isolated from cells 5 produced in step c with a quantifiable compound known to bind to the receptors, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable 10 compound becomes displaced from the receptor.
The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding modified receptors or which modulate the function of modified receptor protein. Compounds which modulate these 15 activities may be DNA,RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding modified receptor, or the function of modified receptor protein.
Compounds that modulate the expression of DNA or RNA encoding 20 modified receptor or the function of modified receptor protein may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression 25 or function in a standard sample.
Kits cont~inin.~ modified receptor DNA, antibodies to modified receptor, or modified receptor protein may be prepared.
Such kits are used to detect DNA which hybridizes to modif1ed receptor DNA or to detect the presence of modified receptor protein 30 or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic, taxonomic or epidemiological studies.
The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen CA 02204491 1997-0~-0~
and measure levels of modified receptor DNA, modified receptor RNA or modified receptor protein. The recombinant proteins, DNA
molecules, RNA molecules and antibodies lend themselves to the forrnnl~tion of kits suitable for the detection and typing of modified 5 receptor. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant modified receptor protein or anti-modified receptor antibodies suitable for detecting modified receptor. The carrier may also 10 contain a means for detection such as labeled antigen or enzyme substrates or the like.
Ph~rm~ceutically useful compositions comprising modulators of modified receptor activity, may be form~ ted according to known methods such as by the admixture of a 15 pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Ph~rm~ceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective ~lrnini~tration~ such compositions will contain an effective amount of the protein, DNA, RNA, or 20 modulator.
Therapeutic or diagnostic compositions of the invention are ~lmini~tered to an individual in amounts sufflcient to treat or diagnose disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and 25 age. Other factors include the mode of ~lmini.~tration.
The ph~rrn~ceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
The term "chemical derivative" describes a molecule 30 that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may ~ttenll~te undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Exarnples of such CA 02204491 1997-0~-0~
moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co-5 ~lmini~tration or sequential ~dmini~tration of other agents may bedesirable.
The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present 10 invention. The compositions containing compounds identified according to this invention as the active ingredient can be ~(lmini~tered in a wide variety of therapeutic dosage forms in conventional vehicles for ~lmini~tration. For example, the compounds can be ~lmini~tered in such oral dosage forms as tablets, 15 capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspenslons, syrups and emulsions, or by injection. Likewise, they may also be ~tlmini~tered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or 20 intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
Advantageously, compounds of the present invention may be ~lmini~tered in a single daily dose, or the total daily dosage may be ~clmini~tered in divided doses of two, three or four times 25 daily. Furthermore, compounds for the present invention can be ~clmini~tered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be ~dmini.~tered in the form of a transdermal delivery system, the 30 dosage a-lmini~tration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, -CA 02204491 1997-0~-0~
the active agents can be ~lmini~tered concurrently, or they each can be ~-lmini~tered at separately staggered times.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors 5 including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of ~clmini~tration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective 10 amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the 15 distribution, equilibrium, and elimin~tion of a drug.
The modified G-protein coupled receptors of the present invention are exemplified herein by the neuropeptide Y receptors.
Deletion mutagenesis of the ~2-adrenergic receptor has shown that none of the hydrophobic clusters of amino acids (the putative 20 transmembrane helices) could be deleted without substantial loss of binding. In contrast, most of the connecting loops could be deleted without affecting the ligand binding properties of the receptor. This indicates that these hydrophilic loops are not required for ligand binding to the receptor, suggesting that the ligand binding pocket is 25 located predomin~ntly within the transmembrane domain of the protein (Strader, et al. FASEB J .3: 182-183 (1989)). Deletions in the connecting loops that were large enough to encompass the entire loop led to steric problems, resulting in incorrect processing of the protein (Dixon, et al. EMBO J. 6: 3269-3275 (1987)). Certain connecting loop 30 deletion mutations, however, led to loss of functional activation of adenylyl cyclase by the receptor. For example, deletion of the carboxy terminal region of the third intracellular loop attenuated the ability of the receptor to activate adenylyl cyclase, and deletion of the amino terminal portion of this loop abolished adenylyl cyclase activation CA 02204491 1997-0~-0~
(Strader, et al. J. Biol. Chem. 262: 16439-16443 (1987)). Moreover, the agonist binding isotherms for these modified receptors displayed a single affinity site, suggesting altered G protein interactions. Since these modified receptors also retain their functional activation of Na+-5 H+ exchange, which is mediated through a different G protein (Barber,et al. Mol. Pharm. 41: 1056-1060 (1992)), the deletions appear not to result in gross structural perturbations of the receptor, suggesting that the changes seen in adenylyl cyclase activation are due to alteration of a specific G protein interaction. Subsequent amino acid replacements in 10 the third intracellular loop confirmed the role of this region in G
protein interaction (Cheung, et al. Mol. Pharm. 41: 1061 - 1065 (1992)).
Modified NPY1 receptors lacking between 6 and 12 amino acids in the N termin~l portion of the third intracellular loop (connecting transmembrane helices 5 and 6) may be synthesized. The 15 bottom of transmembrane helix 5 is defined by the presence of a charged amino acid (human NPY1 Lys233, rat NPYl Lys232) at the end of a series of hydrophobic amino acids. The modified receptors include the deletion of 6-12 residues following Lys233 (human) or Lys232 (rat) (i.e., I234YIRLKRRNNMM; Seq. I.D. No. 1).
20 Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., K238, R239 or R240), or replacement of such residues with alanine or a helix-disrupting residue such as proline.
A second group of modified NPY1 receptors encompass the 25 deletion of 6-13 residues at the C termin~l end of the third intracellular loop of the receptor. The C terminus of this loop is defined by the bottom of helix 6, defined by the presence of the charged residue Arg260 (human NPY1) or Arg259 (rat NPY1) preceding a stretch of hydrophobic amino acids. The modified receptors of this group have 30 deletions of 6-13 residues preceding Arg 260 in hllm~n NPY1 (i.e., KMRDNKYRSSETK259; Seq. I.D. No. 2) and proceeding Arg259 in rat NPY1 (i.e., KIRDSKYRSSETK258; Seq. I.D. No. 4). Alternatively, this sequence could be disrupted by deletion of one or more of the CA 02204491 1997-0~-0~
WO 96/14331 PCI'/US95/14377 charged residues (ie., R249, D250, K252), or replacement of such residues with ~l~nine or a helix-disrupting residue such as proline.
Modified NPY2 receptors lacking between 6 and 12 amino acids in the N terminal portion of the third intracellular loop 5 (connecting transmembrane helices 5 and 6) may be synthesized. The bottom of transmembrane helix 5 is defined by the presence of a charged amino acid (Arg241 in rat and hl1m~n) at the end of a series of hydrophobic amino acids. The modified receptors include the deletion of 6-12 residues following Arg241 (i.e., I242WSKLKNHVSPG; Seq.
10 I.D. No. 5). Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., K244, K246 or H248), or replacement of such residues with ~l~nine or a helix-disrupting residue such as proline.
A second group of modified NPY2 receptors encompass the 15 deletion of 6-13 residues at the C terminal end of the third intracellular loop of the receptor. The C terminus of this loop is defined by the bottom of helix 6, defined by the presence of the charged residue (Lys268 in human and rat) preceding a stretch of hydrophobic amino acids. The modified receptors of this group have deletions of 6-13 20 residues preceding Lys268 in human NPY2 (i.e., ANDHYHQRRQK1~267; Seq. I.D. No. 6) and proceeding Lys268 in rat NPY2 (i.e., AASDHYHQRRHKl-r267; Seq. I.D. No. 7). Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., D257, H258, H260, R262, R263, H264, K265), or 25 replacement of such residues with ~l~nine or a helix-disrupting residue such as proline.
Modified NPY4 receptors lacking between 6 and 12 amino acids in the N terminal portion of the third intracellular loop (connecting transmembrane helices 5 and 6) may be synthesized. The 30 bottom of transmembrane helix 5 is defined by the presence of a charged amino acid (Arg236 in rat and human) at the end of a series of hydrophobic amino acids. The modified receptors include the deletion of 6-12 residues following Arg236 (i.e., I237YRRLQRQGRVF in hllm~n NPY4 (Seq. I.D. No. 8) and I237YQRLQRQRRAF in rat NPY4 CA 02204491 1997-0~-0~
W0 96/14331 ~C~IUS95/14377 (Seq. I.D. No. 9)). Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., R238, R239, R242, R245), or replacement of such residues with ~l~nine or a helix-dis~ g residue such as proline.
A second group of modified NPY4 receptors encompass the deletion of 6-13 residues at the C terrnin~l end of the third intracellular loop of the receptor. The C terminus of this loop is defined by the bottom of helix 6, defined by the presence of the charged residue (Gln263 in hllm~n and Arg263 in rat) preceding a stretch of hydrophobic amino acids. The modified receptors of this group have deletions of 6-13 residues preceding Gln263 in human NPY4 (i.e., HKGTYSLRAGHMK263; Seq. I.D. No. 10) and proceeding Arg263 in rat NPY4 (i.e., HTHTCSSRVGQMK263; Seq. I.D. No. 11).
Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., H251, K252, R258, H261 ), or replacement of such residues with ~l~nine or a helix-di~ g residue such as proline.
Other modified receptors encompass the deletion of 6-13 residues at either the N or C terminal end of the third intracellular loop, or replacement of residues within this region, of other members of the family of NPY receptors. The N terminus of the third intracellular loop (connecting transmembrane helices 5 and 6) is defined by the presence of a charged or polar amino acid at the end of the fifth series of hydrophobic amino acids in the sequence of the receptor (helix 5).
The C te~nimls of this loop is located at the bottom of helix 6, defined by the presence of a charged or polar residue preceding the sixth stretch of hydrophobic amino acids.
Other modified receptors encompass the addition of 5 to 10 residues at either the N or C terminal end of the third intracellular loop of the NPY1, 2 or 4 receptors such that the amphipathic nature of these regions is disrupted.
The following examples are provided to further define the invention without, however, limiting the invention to the particulars of the examples.
CA 02204491 1997-0~-05 W O96114331 PCTrUS95/14377 Deletion of 6-13 amino acids at the N-terminal portion of the third 5 intracellular loop of the hllm~n Neuropeptide Y1 receptor Modified receptor is constructed by site-directed mutagenesis of the human neuropeptide Y1 receptor cDNA by standard molecular biological techniques.
The modified DNA sequence encodes a hllm~n neuropepide 10 Yl receptor lacking between 6 and 13 amino acid residues at the N-terminal portion of the third intracellular loop. The nucleotide sequence of the modified receptor is confirmed by DNA sequencing~
As with modified ~2 receptors, the modified NPY receptor is designed so as to dismpt the proximal portion of the third intracellular loop, 15 without affecting the adjacent fifth transmembrane helix. Thus, the charged amino acid that delineates the bottom of helix 5 (Lys233) is left intact in the modified receptor, while the six to thirteen amino acids which follow it are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, beginning 20 immediately after the charged residue at the bottom of transmembrane helix 5, for example D(234-241)NPYl receptor.
25 Deletion of 6-13 amino acids at the C-terminal portion of the third intracellular loop of the hllm~n Neuropeptide Y1 receptor Modified human NPY1 receptor, lacking 13 residues at the C-terminal portion of the third intracellular loop (D(247-259)NPYI
receptor), is prepared by standard mutagenesis procedures. The 30 nucleotide sequences of the modified receptors are confirmed by DNA
sequencing. This modified human NPY1 receptor is designed so as to disrupt the distal portion of the third intracellular loop, without affecting the adjacent sixth transmembrane helix. Thus, the charged amino acid that defines the bottom of helix 6 (Lys260) is left intact, CA 02204491 1997-0~-0~
while the nearby proximal residues are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, ending immediately before the charged residues at the bottom of helix 6.
s Expression and characterization of the altered Neuropeptide Y1 receptor COS-7 cells are transfected with the modified receptor cDNA subcloned into a eukaryotic expression vector such as the eukaryotic expression vector pcDNA I/neo (Invitrogen). Cells are harvested after incubation for about 60-72 h. Membranes cont~ining the expressed receptor protein are prepared as described (C. D. Strader et al., Proc. Natl. Acad. Sci. U.S.A. 84, 4384-4388 (1987).
Binding reactions are performed in a final volume of 250 ,ul of buffer A (50 ,uM Tris, pH 7.4 cont~ining 20 mM CaC12, 5 mM
KCI, 0.2% bovine serum albumin, 10 ,uM phosphoramidon, 40 ~lg/ml bacitracin and 2 ,ug/ml leupeptin). 125I-NPY or 125I-PYY (0.1 nM) is incubated with membranes for 2 hr at 25~C before filtration over GF/C
filters presoaked in 0.1% polyethyleneimine. Filters are washed with ice cold buffer A before analysis of the bound radioactivity by scintill~tion counting.
Membranes prepared from the COS-7 cells transfected with a vector cont~ining either the wild type or the modified receptor cDNA
specifically bind a radiolabeled neuropeptide Y receptor radioligand.
The modified receptor is characterized by an absence of coupling to G
proteins, an inability to mediate the activation of second messenger systems, and an increased affinity for agonists.
The modified neuropeptide Y receptor, when expressed in m~mm~ n cells, does not stimulate G protein activation in response to the agonist NPY. In contrast, when the wild type receptor is expressed in the same cell line, activity is stimulated.
CA 02204491 1997-0~-0~
~ WO 96tl4331 PCT/US95/14377 These modified receptors have increased affinity for agonists when compared to the wild type receptor. The wild type NPYl receptor can be described pharmacologically by the relative potency of peptide ligands: neuropeptide Y= peptide YY > [Leu31Pro34]NPY >
5 NPY[2-36]>> NPY[13-36], with the affinity of NPY in the range of 0.1-10 nM, and NPY[13-36] having an affinity in the ~M range. The mutant receptor binds the agonists with the same relative order of potency. The high affinity of the agonist for the modified receptor is not affected by agents that uncouple the receptor from the G protein;
10 such agents include the nonhydrolyzable GTP analog GppNHp, sodium fluoride, and the detergent digitonin. In contrast, the wild type receptor binds agonists with two affinity states: a high affinity state, indicative of binding to the receptor-G protein complex, and a low affinity state, reflecting binding to the uncoupled receptor alone. When the receptor 15 is not optimally coupled to the G protein, a binding assay using the modified receptor will detect agonists with more sensitivity than will the identical binding assay using the wild-type receptor.
Screening Assay usin~ modified Neuropeptide Y1 receptors Transfected cells expressing recombinant modified receptor may be used to identify compounds that bind to the receptor with high affinity. This may be accomplished in a variety of ways, such as by 25 incubating the test compound in a final volume of 0.25 ml of buffer A
with membranes cont~ining 5-7 pM of the modified neuropeptide Y
receptor and 100 pM 125I-PYY or 125I-NPY for 2 hour at 25~. The reaction is stopped by filtration over GF/C glass fiber filters presoaked in 0.1% polyethyleneimine, washing with 3 x 5 ml of cold buffer A, and 30 counting the filters in a gamma counter to measure bound radioactivity.
This assay will detect a compound that has a high intrinsic affinity for the receptor. Such compounds may be either agonists or antagonists.
CA 02204491 1997-0~-0~
Deletion of 6-13 amino acids at the N-terrnin~l portion of the third intracellular loop of Neuropeptide Y receptor subtypes Modified NPY receptor subtypes (e.g., NPY2, NPY4) having deletions at the N terminal region of the third intracellular loop are constructed by site-directed mutagenesis of the neuropeptide Y
receptor cDNA by standard molecular biological techniques.
The modified DNA sequence encodes a neuropepide Y
receptor lacking between 6 and 13 amino acid residues at the N-terminal portion of the third intracellular loop. The nucleotide sequence of the modified receptor is confirmed by DNA sequencing. The modified NPY receptor is designed so as to disrupt the proximal portion of the third intracellular loop, without affecting the adjacent fifth transmembrane helix. Thus, the charged amino acid that delineates the bottom of helix 5 is left intact in the modified receptor, while the six to thirteen amino acids which follow it are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, be~innin~ imrnediately after the charged residue at the bottom of transmembrane helix 5.
Deletion of 6-13 amino acids at the C-terminal portion of the third intracellular loop of the Neuropeptide Y receptor Modified NPY receptor subtypes (e.g., NPY2, NPY4) having deletions at the C termin~l region of the third intracellular loop are constructed by site-directed mutagenesis of the neuropeptide Y
receptor cDNA by standard molecular biological techniques. The nucleotide sequences of the modified receptors are confirmed by DNA
sequencing. These modified NPY receptors have disruptions in the distal portion of the third intracellular loop, without affecting the adjacent sixth transmembrane helix. Thus, the polar amino acid that defines the bottom of helix 6 is left intact, while the nearby proximal CA 02204491 1997-0~-0~
WO 96/14331 PCI'IUS95/14377 residues are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, ending immediately before the polar residues at the bottom of helix 6.
Expression and characterization of modified NPY Receptor The modified receptor is subcloned into an expression vector such as pRC/CMV (Invitrogen,San Diego, CA) and expressed in 10 m~mm~ n cells bytransfection. Approximately 72hours after transfection, cells are harvested for radioligand binding assays.
For binding assays, the membranes are prepared by harvesting the cells in ice-cold lysis buffer (5 mg Tris, pH 7.4; 2 mM
EDTA), followed by 15 min centrifugation at 38,000 x g. The 15 membrane pellet is then resuspended in buffer A. Equilibrium binding to the wild type or modified NPY receptor is performed in a final volume of 0.25 ml cont~ining membranes, 100 pM 125I-PYY, and serial dilution of the competing ligands. Binding reactions are incubated for 2 hr at 25~C, and terminated by rapid filtration over 20 GF/C filters pre-soaked in 0.1% polyethyleneimine. The radioactivity is quantified with a Packard gamma counter.
These modified receptors have increased affinity for agonists when compared to the wild type receptor. The wild type "atypical NPY1" or NPY4 receptor that mediates feeding behavior can 25 be described phar~nacologically by the high affinity of neuropeptide Y, peptide YY, NPY[2-36], and [Leu31Pro34]NPY, and the lower affinity of more truncated analogs NPY[13-36] and NPY [20-36], and structurally by its sequence homology (>45% at the DNA level) to the NPY1 receptor. The affinity of NPY for the atypical Y1 receptor 30 subtype is in the range of 0.01-10 nM, and that for NPY[13-36] is in the 0.1-10 ~M range. The mutant receptor binds the agonists with the same relative order of potency as the wild type receptor. The high affinity of the agonist for the modified receptor is not affected by agents that uncouple the receptor from the G protein; such agents include the 35 nonhydrolyzable GTP analog GppNHp, sodium fluoride, and the CA 02204491 1997-0~-05 detergent digitonin. In contrast, the wild type receptor binds agonists with two affinity states: a high affinity state, indicative of binding to the receptor-G protein complex, and a low affinity state, reflecting binding to the uncoupled receptor alone. When the receptor is not optimally S coupled to the G protein, a binding assay using the modified receptor will detect agonists with more sensitivity than will the identical binding assay using the wild-type receptor. Other NPY receptor subtypes (NPY2, NPY3, and others) are also defined pharmacologically by the relative potencies of peptide ligands for these receptors and str~cturally 10 by their sequence similarity to the NPY 1 receptor. Mutant receptors having deletions in the third intracellular loop have similar orders of potency as the corresponding wild type receptor, but with higher affinity than the wild type receptor in the absence of G protein coupling.
These modified NPY receptors are readily used in a screening assay to detect compounds that bind with high affinity to the NPY receptor subtype, regardless of whether these compounds are agonists or antagonists.
Cloning and Expression of Modified NPY Receptor cDNA into Bacterial Expression Vectors Recombinant modified receptor is produced in a bacterial expression system such as E. coli. The modified receptor expression 25 cassette is transferred into an E. coli expression vector; expression vectors include but are not limited to, the pET series (Novagen). The pET vectors place modified receptor expression under control of the tightly regulated bacteriophage T7 promoter. Following transfer of this construct into an E. coli host which contains a chromosomal copy of the 30 T7 RNA polymerase gene driven by the inducible lac promoter, expression of modified receptor is induced by addition of an approl,liate lac substrate (IPTG) is added to the culture. The levels of expressed modified receptor are determined by the assays described herein.
CA 02204491 1997-0~-0~
Cloning and Expression of Modified NPY Receptor cDNA into a Vector for Expression in Insect Cells Baculovirus vectors derived from the genome of the AcNPV virus are designed to provide high level expression of cDNA in the Sf9 line of insect cells (ATCC CRL# 1711). Recombinant baculovirus expressing modified receptor cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the modified receptor cDNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18, 5667 (1990)] into Sf9 cells. Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of ~-galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, modified receptor expression is measured.
Authentic modified receptor is found in association with the infected cells. Active modified receptor is extracted from infected cells by hypotonic or detergent lysis.
Alternatively, the modified receptor is expressed in the Drosophila Schneider 2 cell line by cotransfection of the Schneider 2 cells with a vector cont~ining the modified receptor DNA downstream and under control of an inducible metallothionin promoter, and a vector encoding the G418 resistant neomycin gene. Following growth in the presence of G418, resistant cells are obtained and induced to express modified receptor by the addition of CuSO4. Identification of modulators of the modified receptor is accomplished by assays using either whole cells or membrane preparations.
CA 02204491 1997-0~-0~
WO 96/14331 PCI~/US9~/14377 Cloning of Modified NPY Receptor cDNA into a yeast expression vector S Recombinant modified receptor is produced in the yeast S.
cerevisiae following the insertion of the modified receptor cDNA
cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins. In the case of intracellular expression, vectors such as EmBLyex4 or the like are ligated to the modified receptor cistron [Rinas, U. et al., Biotechnology 8, 543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265, 4189-4192 (1989)]. For extracellular expression, the modified receptor cistron is ligated into yeast expression vectors which fuse a secretion signal. The levels of expressed modified receptor are determined by the assays described herein.
Purification of Recombinant Modified NPY Receptor Recombinantly produced modified receptor may be purified by a variety of procedures, including but not limited to antibody affinity chromatography.
Modified receptor antibody affinity columns are made by adding the anti-modified receptor antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. Thè rem~ining activated esters are then quenched with 1 M ethanolamine HCI (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents, and the cell culture supernatants or cell extracts cont~ining solubilized modified receptor or modified CA 02204491 1997-0~-0~
WO 96/14331 PCI/US9Stl4377 receptor subunits are slowly passed through the column. The column is then washed with phosphate-buffered saline (PBS) supplemented with detergents until the optical density (A280) falls to background; then the protein is eluted with 0.23 M glycine-HCI (pH 2.6) supplemented with S detergents. The purified modified receptor protein is then dialyzed against PBS.
10 Cloning and Expression of Modified NPY Receptor in M~mm~ n Cell System A modified receptor is cloned into a m~mm~ n expression vector. The m~mm~ n expression vector is used to transform a m~mm~ n cell line to produce a recombinant m~mm~ n cell line.
15 The recombinant m~mm~ n cell line is cultivated under conditions that permit expression of the modified receptor. The recombinant m~mm~ n cell line or membranes isolated from the recombinant m~mm~lian cell line are used in assays to identify compounds that bind to the modified receptor.
Screening Assay Recombinant cells co-lt~ DNA encoding a modified 25 NPY receptor, membranes derived from the recombinant cells, or recombinant modified receptor preparations derived from the cells or membranes may be used to identify compounds that modulate modified NPY receptor activity. Modulation of such activity may occur at the level of DNA, RNA, protein or combinations thereof. One method of 30 identifying compounds that modulate modified NPY receptor, comprises:
(a) mixing a test compound with a solution cont~ining modified NPY receptor to form a mixture;
CA 02204491 1997-05-0~
WO 96114331 PCT/US95tl4377 (b) measuring modified NPY receptor activity in the mixture; and (c) comparing the modified NPY receptor activity of the mixture to a standard.
Formulation of Pharmaceutical Compositions Compounds identified by the method of Example 13 are 10 formulated into pharmaceutical compositions according to standard method.s. The compounds or pharmaceutical compositions are used either alone or in combination with other compounds or compositions for the treatment of ~nim~l.s (including humans) in need of treatment.
Conditions requiring treatment include but are not limited to obesity, 15 regulation of apetite, congestive heart failure, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's dlseases.
Methods of Treatment ~nim~ (including hllm~ns) having a condition, the 25 condition being characterized by factors selected from altered levels of neuropeptide Y, altered activities of neuropeptide Y, altered levels of neuropeptide Y receptor activity, altered neuropeptide Y receptor activity, and combinations thereof, are treated with compounds or derivatives of compounds identified by the screening method or 30 pharmaceutical compositions comprising the compounds or derivative.s of compounds identified by the screening method.
~ nim~l.s (including humans) having a condition selected from obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac vasospasm, CA 02204491 1997-0~-0~
cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, Huntington's Disease, Alzheimer's Disease, Parkinson's disease, and combinations thereof, are treated with a therapeutically effective amount of compounds or derivatives of compounds identified by the 5 screening method or pharmaceutical compositions comprising the compounds or derivatives of compounds identif1ed by the screening method.
While the foregoing specification teaches the principles of 10 the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptions, or modifications, as come within the scope of the following claims and its equivalents.
CA 0220449l 1997-0~-0~
W O 96/14331 PCTrUS95/14377 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: STRADER, CATHERINE D.
CASCIERI, MARGARET A.
MACNEIL, DOUGLAS J.
(ii) TITLE OF INVENTION: MODIFIED NEUROPEPTIDE Y RECEPTORS
(iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MARY A. APPOLLINA
(B) STREET: 126 EAST LINCOLN AVENUE
(C) CITY: RAHWAY
(D) STATE: NEW JERSEY
(E) COUNTRY: US
(F) ZIP: 07065-0900 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/335,017 (B) FILING DATE: 07-NOV-1994 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: APPOLLINA, MARY A.
(B) REGISTRATION NUMBER: 34,087 (C) REFERENCE/DOCKET NUMBER: 19339Y PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908) 594-3462 (B) TELEFAX: (908) 594-4720 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 0220449l l997-0~-0~
W O 96/14331 PCTrUS95/14377 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ile Tyr Ile Arg Leu Lys Arg Arg Asn Asn Met Met (2) INFORMATION FOR SEQ ID NO:2:
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Met Asn Ser Thr Leu Phe Ser Gln Val Glu Asn His Ser Asp Phe Leu Val His Ser Asn Phe Ser Glu Lys Asn Ala Gln Leu Leu Ala Phe Glu Asn Asp Asp Cys His Leu Pro Leu Ala Met Ile Phe Thr Leu Ala Leu Ala Tyr Gly Ala Val Ile Ile Leu Gly Val Ser Gly Asn Leu Ala Leu Ile Ile Ile Ile Leu Lys Gln Lys Glu Met Arg Asn Val Thr Asn Ile Leu Ile Val Asn Leu Ser Phe Ser Asp Leu Leu Val Ala Ile Met Cys Leu Pro Leu Thr Phe Val Tyr Thr Leu Met Asp His Trp Val Phe Gly CA 0220449l l997-0~-0~
Glu Ala Met Cys Lys Leu Asn Pro Phe Val Gln Cys Val Ser Ile Thr Val Ser Ile Phe Ser Leu Val Leu Ile Ala Val Glu Arg His Gln Leu Ile Ile Asn Pro Arg Gly Trp Arg Pro Asn Asn Arg His Ala Tyr Val ~ly Ile Ala Val Ile Trp Val Leu Ala Val Ala Ser Ser Leu Pro Phe ~eu Ile Tyr Gln Val Met Thr Asp Glu Pro Phe Gln Asn Val Thr Leu Asp Ala Tyr Lys Asp Lys Tyr Val Cys Phe Asp Gln Phe Pro Ser Asp Ser His Arg Leu Ser Tyr Thr Thr Leu Leu Leu Val Leu Gln Tyr Phe Gly Pro Leu Cys Phe Ile Phe Ile Cys Tyr Phe Lys Ile Tyr Ile Arg ~eu Lys Arg Arg Asn Asn Met Met Asp Lys Ser Glu Gly Arg Phe His ~er Pro Asn Leu Gly Gln Val Glu Gln Asp Gly Arg Ser Gly His Gly Leu Met Arg Asp Asn Lys Tyr Arg Ser Ser Glu Thr Lys Arg Ile Asn Ile Met Leu Leu Ser Ile Val Val Ala Phe Ala Val Cys Trp Leu Pro Leu Thr Ile Phe Asn Thr Val Phe Asp Trp Asn His Gln Ile Ile Ala ~hr Cys Asn His Asn Leu Leu Phe Leu Leu Cys His Leu Thr Ala Met ~le Ser Thr Cys Val Asn Pro Ile Phe Tyr Gly Phe Leu Asn Lys Asn Phe Gln Arg Asp Leu Gln Phe Phe Phe Asn Phe Cys Asp Phe Arg Ser Arg Asp Asp Asp Tyr Glu Thr Ile Ala Met Ser Thr Met His Thr Asp Val Ser Lys Thr Ser Leu Lys Gln Ala Ser Pro Val Ala Phe Lys Lys CA 02204491 1997-0~-0~
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
His Lys Gly Thr Tyr Ser Leu Arg Ala Gly His Met Lys (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
His Thr His Thr Cys Ser Ser Arg Val Gly Gln Met Lys
21. Grundemar, L., C. Wahlestedt, a nd D.J. Reis, Long-lasting 0 inhibition of the cardiovascular responses to glutamate and the baroreceptor reflex elicited by neurop~ptide Y injected into the nucleus tractus solitarius of the rat. Neurosci Lett, 1991. 122(1): p. 135-9.
22. Zukowska-Grojec, Z. and C. Wallestedt, Origin and actions of 15 neuropeptide Y in the cardiovascular ~ystem, in Neuropeptide Y, W.F.
Colmers and C. Wahlestedt, Editor. 1993, Hllm~n~ Press: Totowa, NJ.
p. 315-388.
23. Edvinsson, L., et al., Congestive heart failure: involvement of 20 perivascular peptides reflecting activi~ in sympathetic, parasympathetic and afferentfibres. Eur J Clin Invest, 990. 20(1): p. 85-9.
24. Franco, C.A., et al., Release of nPuropeptide Y and noradrenaline from the human heart after aortic occlusion during coronary artery 25 surgery. Cardiovasc Res, 1990. 24(3): ?. 242-6.
25. Maisel, A.S., et al., Elevation of ~lasma neuropeptide Y levels in congestive heartfailure. Am J Med, lC89. 86(1): p. 43-8.
30 26. Wahlestedt, C. and D.J. Reis, Neuropeptide Y-related peptides and their receptors - are the receptors potential therapeutic drug targets? Annu. Rev. Ph~ col. Toxicol., 1993. 32: p. 309-352.
27. Wahlestedt, C., S. Regun~th~n, and D.J. Reis, Identification of 35 cultured cells selectively expressing Y1-, Y2-, or Y3-type receptors for neuropeptide Y/peptide YY. Life Sciences, 1992. 50: p. PL7-PL12.
28. Feth, F., W. Rascher, and M.C. Michel, G-protein coupling and signalling of Y1-like neuropeptide Y receptors in SK-N-MC cells.
40 Naunyn Schmiedebergs Arch Pharmaccl, 1991. 344(1): p. 1-7.
CA 02204491 1997~ -0~
29. Motulsky, H.J. and M.C. Michel. Neuropeptide Y mobilizes Ca2+
and inhibits adenylate cyclase in human erythroleukemia cells. Am J
Physiol, 1988. 255: p. E880-E885.
5 30. Wahlestedt, C., et al., Neuropeptide Y receptor subtypes, Yl and Y2. Ann N Y Acad Sci, 1990. 611(7): p. 7-26.
31. Sahu, A. and S.P. Kalra, Neurop~ptidergic regulation of feeding-behavior - neuropeptide-Y. Trends In Endocrinology And Metabolism, 10 1993. 4(7): p. 217-224.
32. Clark, J.T., et al., Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology, 1984.
115(1): p. 427-429.
33. Stanley, B.G. and S.F. Leibowitz, Neuropeptide Y injected in the paraventricular hypothalamus: a powerful stimulant offeeding behavior. Proc. Natl. Acad. Sci. USA, 1985. 82: p. 3940-3943.
20 34. Stanley, B.G. and S.F. Leibowitz, Neuropeptide Y: stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci, 1984. 35(26): p. 2635-42.
35. Zarjevski, N., et al., Chronic intracerebroventricular 25 neuropeptide-Y administration to normal rats mimics hormonal and metabolic changes of obesity. Endocrinology, 1993. 133(4): p. 1753-1758.
i 36. Billington, C.J. and A.S. Levine, Hypothalamic neuropeptide Y
30 regulation offeeding and energy metabolism. Current Opinion in Neurobiology, 1992. 2: p. 847-851.
37. Leibowitz, S.F., Brain neuropeptide Y: an integrator of endocrine, metabolic and behavioral processes. Brain Research Bulletin, 35 1991. 27: p. 333-337.
38. Billington, C.J., et al., Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am. J. Physiol., 1991. 260:
p. R321-R327.
CA 02204491 1997-0~-0~
WO 96/14331 PCI'/US95/14377 39. Billington, C.J., et al., Neuropeptide-Y in hypothalamic paraventricular nucleus - a center coordinating energy-metabolism.
American Journal Of Physiology, 1994. 266(6): p. R1765-R1770.
5 40. Kalra, S.P., et al., Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food.
Proc. Natl. Acad. Sci. USA, 1991. 88: p. 10931-10935.
41. Beck, B., et al., Rapid and localized alterations of neuropeptide Y
0 in discrete hypothalamic nuclei with feeding status. Brain Res, 1990.
528(2): p. 245-9.
42. Brady, L.S., et al., Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats.
15 Neuroendocrinology, 1990. 52(5): p. 441-7.
43. Calza, L., et al., Increase of neuropeptide Y-like immunoreactivity in the paraventricular nucleus offasting rats.
Neurosci Lett, 1989. 104(1-2): p. 99-104.
44. Sahu, A., P.S. Kalra, and S.P. Kalra, Food deprivation and ingestion induce reciprocal changes in neuropeptide Y concentrations in the paraventricular nucleus. Peptides, 1988. 9(1): p. 83-6.
25 45. Abe, M., et al., Increased neuropeptide Y content in the arcuato-paraventricular hypothalamic neuronal system in both insulin-dependent and non-insulin-dependent diabetic rats. Brain Res, 1991. 539(2): p.
223-7.
30 46. Sahu, A., et al., Neuropeptide-Y concentration in microdissected hypothalamic regions and in vitro release from the medial basal hypothalamus-preoptic area of streptozotocin-diabetic rats with and without insulin substitution therapy. Endocrinology, 1990. 126(1): p.
192-8.
47. White, J.D., et al., Increased hypothalamic content of preproneuropeptide-Y messenger ribonucleic acid in streptozotocin-diabetic rats. Endocrinology, 1990. 126(2): p. 765-72.
40 48. Willi~m.~, G., et al., Increased hypothalamic neuropeptide Y
concentrations in diabetic rat. Diabetes, 1988. 37(6): p. 763-72.
CA 02204491 1997-o~-o~
49. Willi~m.~, G., et al., Increased neuropeptide Y concentrations in specific hypothalamic regions of streptozocin-induced diabetic rats.
Diabetes, 1989. 38(3): p. 321-7.
50. Beck, B., et al., Hypothalamic neuropeptide Y (NPY) in obese Zucker rats: implications in feeding and sexual behaviors. Physiol Behav, 1990. 47(3): p. 449-53.
10 51. Sanacora, G., et al., Increased hypothalamic content of preproneuropeptide Y messenger ribonucleic acid in genetically obese Zucker rats and its regulation by food deprivation. Endocrinology, 1990. 127(2): p. 730-7.
15 52. Wahlestedt, C., R. F.km~n, and E. Widerlov, Neuropeptide Y
(NPY) and the central nervous system: distribution effects and possible relationship to neurological and psychiatric disorders. Prog Neuropsychoph~ col Biol Psychiatry, 1989. 13(1-2): p. 31-54.
20 53. Larh~mm~r, D., et al., Cloning and functional expression of a human neuropeptide Y/peptide YY receptor of the Yl-type. J. Biol.
Chem., 1992. 267: p. 10935- 10938.
54. Sheikh, S.P., et al., Localization of Y] receptors for NPY and 25 Pn on vascular smooth muscle cells in rat pancreas. Arn J Physiol, 1991. 260: p. G250-G257.
55. Wahlestedt, C., N. Y~n~ih~ra, and R. Hakanson, Evidence for diJ~ferent pre-and post-junctional receptors for neuropeptide Y and 30 related peptides. Regul Pept, 1986. 13(3-4): p. 307-18.
56. Jorgensen, J.C., J. Fuhlendorff, and T.W. Schwartz, Structure-function studies on neuropeptide Y and pancreatic polypeptide--evidence for two PP-fold receptors in vas deferens. Eur J Ph~ Gol, 1990.
35 186(1): p. 105-14.
57. Cox, H.M. and J.L. Krstenansky, The effects of selective amino acid substitution upon neuropeptide Y antisecretory potency in rat jejunum mucosa. Peptides, 1991. 12(2): p. 323-7.
CA 02204491 1997-0~-0~
58. Aicher, S.A., et al., Receptor-selective analogs demonstrate NPYIPYY receptor heterogeneity in rat brain. Neurosci Lett, 1991.
130(1): p. 32-6.
5 59. Balasubr2m~ni~m, A., et al., Characterization of neuropeptide Y
binding sites in rat cardiac ventricular membranes. Peptides, l 990.
11(3): p. 545-50.
60. Li, X.J., et al., Cloning, functional expression, and developmental 0 regulation of a neuropeptide Y receptor from Drosophila melanogaster.
J Biol Chem, 1992. 267(1): p. 9-12.
61. Roman, F.J., et al., Neuropeptide Y and peptide YY interact with rat brain sigma and PCP binding sites. Eur J Pharmacol, 1989. 174(2-15 3): p. 301-2.
62. Schwartz, T.W., S.P. Sheikh, and M.M. O'Hare, Receptors on phaeochromocytoma cells for two members of the PP-fold family--NPY
and PP. Febs Lett, 1987. 225(1-2): p. 209-14.
63. Schwartz, T.W., et al., Signal epitopes in the three-dimensional structure of neuropeptide Y. Interaction with Yl, Y2, and pancreatic polypeptide receptors. Ann N Y Acad Sci, 1990. 611(35): p. 35-47.
25 64. Wahlestedt, C., et al., Modulation of anxiety and neuropeptide Y-Yl receptors by antisense oligodeoxynucleotides. Science, 1993. 259: p.
528-531.
65. Jolicoeur, F.B., et al., In vivo structure activity study supports the 30 existence of heterogeneous neuropeptide Y receptors. Brain Res Bull, 1991. 26(2): p. 309-11.
66. Leibowitz, S.F. and J.T. Alexander, Analysis of neuropeptide Y-inducedfeeding: dissociation of Yl and Y2 receptor effects on natural 35 meal patterns. Peptides, 1991. 12(6): p. 1251 -60.
67. Inui, A., et al., Characterization of peptide YY receptors in the brain. Endocrinology, 1989. 124(1): p. 402-9.
CA 02204491 1997-0~-0~
WO 96/14331 PCI~/US95/14377 68. Boublik, J., et al., Neuropeptide Y and ne~lropeptide Yl~-36.
Structural and biological characterization. Int J Pept Protein Res, 1989.
33(1): p. 11-5.
5 69. Eva, C., et al., Molecular cloning of a novel G protein-coupled receptor that may belong to the neuropeptide receptor family. FEBS
Lett., 1990. 271: p. 81-84.
70. Herzog, H., et al., Cloned human neuropeptide Y receptor couples 1O to two di~erent second messenger systems. Proc Natl Acad Sci U S A, 1992. 89: p. 5794-5798.
71. Strader, C.D., I.S. Sigal, and R.A. Dixon, Structural basis of beta-adrenergic receptor function. FASEB-J, 1989. 3(7): p. 1825-1832.
72. Bard, J.A., Walker, M.W., Brancheck, T., Wein~h~nk, R. DNA
encoding a human neuropeptide Ylpeptide YYIpancreatic polypeptide receptor (Y4) and uses thereof. PCT International Application Publication No. WO 95/17906, published 6 July 1995.
73. Gerald, C., Walker, M.W., Branchek, T., and Weinshank, R.
Nucleic acid encoding Neuropeptide YlPeptide YY (Y2( receptors and uses thereof. PCT International Application Publication No.
WO95/21245, published 10 August 1995.
74. Rose, P. A. et al., Cloning and functional expression of a cDNA
encoding the human type 2 neuropeptide Y receptor. J. Biol. Chem., 270:22661 -22664.
Modified neuropeptide Y receptors having deletions, replacements or additions in the third intracellular domain are identified and methods of making the modified receptors are provided. The invention includes the modified receptors, assays employing the 35 modified receptors, cells expressing the modified receptors, compounds identified through the use of the modif1ed receptors, including modulators of the receptors, and the use of the compounds to treat conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95114377 cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases.
BREF DESCRIPTION OF THE DRAWINGS
5 Figure 1. Schematic diagram of G-protein signal transduction system.
The receptor is shown as a seven-helical bundle. a, ~, and ~ indicate the three subunits of the G protein. E indicates an effector enzyme, such as adenylyl cyclase. The agonist ~A) binding with high affinity to the receptor-G protein complex and with low affinity to the receptor 10 alone is shown.
Figure 2. Schematic diagram of the hamster ~2 adrenergic receptor.
The third intracellular loop comprises residues 221-273. The proximal and distal segments of this loop are drawn in cylinders.
Figure 3 shows the amino acid sequence of the human NPY1 receptor subtype aligned with that of the hamster ~2-adrenergic receptor. The transmembrane helices are underlined.
Modified neuropeptide Y receptors having deletions, replacements or additions in the third intracellular domain are identified and methods of m~king the modified receptors are provided. The invention includes the modified receptors, assays employing the 25 modified receptors, cells expressing the modified receptors, compounds identified through the use of the modified receptors, including modulators of the receptors, and the use of the compounds to treat conditions, including obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and 30 cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's diseases. Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers.
CA 02204491 1997-0~-0~
WO 96/14331 PCI/US95tl4377 Neuropeptide Y receptors belong to a class of receptors known as "G-protein coupled receptors." The term "G-protein coupled receptor" refers to any receptor protein that mediates its endogenous signal transduction through activation of one or more guanine nucleotide binding regulatory proteins (G-proteins). These receptors share common structural features, including seven hydrophobic transmembrane domains. G-protein coupled receptors include receptors that bind to small biogenic amines, including but not limited to beta-adrenergic receptors (~AR), alpha-adrenergic receptors (ocAR) and muscarinic receptors, as well as receptors whose endogenous ligands are peptides, such as neurokinin, neuropeptide Y and glucagon receptors.
Examples of ~AR include beta-1, beta-2, and beta-3 adrenergic receptors.
G-protein coupled receptors are cell surface proteins that mediate the responses of a cell to a variety of environmental signals.
Upon binding an agonist, the receptor interacts with one or more specific G proteins, which in turn regulate the activities of specific effector proteins. By this means, activation of G-protein coupled receptors amplifies the effects of the environmental signal and initiates a cascade of intracellular events that ultimately leads to defined cellular responses. G-protein coupled receptors function as a complex information processing network within the plasma membrane of the cell, acting to coordinate a cell's response to multiple environmental signals.
G-protein coupled receptors are characterized by the ability of agonists to promote the formation of a high affinity ternary complex between the agonist, the receptor and the G-protein (Figure 1). The a-subunit of the G protein contains a guanine nucleotide binding site which, in the high affinity ternary [G protein-receptor-agonist]
complex, is occupied by GDP. In the presence of physiological concentrations of GTP, the GDP molecule in the guanine nucleotide binding site of the G protein is displaced by a GTP molecule. The binding of GTP dissociates the a subunit of the G protein from its ~ and y subunits and from the receptor, thereby activating the G-protein to CA 02204491 1997-0~-0~
stimulate downstream effectors (adenylyl cyclase in the case of the ,B-adrenergic receptor (~AR)) and propagating the intracellular signal.
Thus, the ternary complex is transient in the presence of physiological concentrations of GTP. Because the affinity of the agonist for the 5 receptor-G protein complex is higher than its affinity for the uncomplexed receptor, one consequence of the destabilization of the ternary complex is a reduction in the affinity of the receptor for the agonist. Thus, the affinity of agonists for G-protein coupled receptors is a function of the efficiency with which the receptor is coupled to the 10 G-protein. In contrast, antagonists bind with the same affinity to the receptor in the presence or absence of G-protein coupling.
The observation that agonist affinity can be reduced by conditions under which a receptor is not optimally coupled to its G-protein has important implications for the identification of agonists of 15 G-protein coupled receptors, particularly identification based on ligand binding. If a receptor is not optimally coupled to the G-protein under the conditions of binding assays, an agonist will bind to the receptor with relatively low affinity. Thus, a screen that relies on a binding assay based on displacement of a radiolabeled ligand, although attractive 20 for its ease and the potential for high throughput, poses the risk that a promising partial agonist might be overlooked because the agonist would bind predominantly to the low affinity state of the receptor, and thus would have low affinity in the binding assay. Consequently, functional assays are frequently used to screen for agonists of G-protein 25 coupled receptors. However, functional assays (ranging from ex vivo muscle contraction assays to determination of second messenger levels in cells expressing exogenous cloned G-protein coupled receptors) are tedious and more time-consuming than ligand binding assays, and hence are not readily adapted to high-throughput screens. Because the 30 modified receptors of the present invention bind agonists with high affinity in the presence or absence of the G-protein, they can be used in high throughput radioligand binding assays to screen for high affinity ligands, regardless of whether the ligands are agonists or antagonists.
CA 02204491 1997-0~-0~
G-protein coupled receptors consist of seven hydrophobic domains connecting eight hydrophilic domains. The hydrophobicity or hydrophilicity of the domains may be determined by standard hydropathy profiles, such as Kyte-Doolittle analysis (Kyte, J. and 5 Doolittle, R.J.F. J. Mol. Biol. 157: 105 (1982)). The receptors are thought to be oriented in the plasma membrane of the cell such that the N-terminus of the receptor faces the extracellular space and the C-terminus of the receptor faces the cytoplasm, so that each of the hydrophobic domains crosses the plasma membrane. The receptors 10 have been modeled and the putative boundaries of the extracellular, transmembrane and intracellular domains are generally agreed (for a review, see Baldwin, EMBO J. 12:1693, 1993). In general, the transmembrane domains are comprised of stretches of 20-25 amino acids in which most of the amino acid residues have hydrophobic side 15 chains (including cysteine, methionine, phenyl~l~nine, tyrosine, tryptophan, proline, glycine, ~l~nine, valine, leucine, isoleucine), whereas the intracellular and extracellular loops are defined by contiguous stretches of several amino acids that have hydrophilic or polar side chains (including aspartate, glutamate, asparagine, glutamine, 20 serine, threonine, histidine, lysine, and arginine). Polar amino acids, especially uncharged ones (such as serine, threonine, asparagine, and gll-t~mine) are found in both transmembrane and extramembrane regions.
The extramembrane regions are characterized by 25 contiguous stretches of three or more hydrophilic residues. In contrast, hydrophilic residues are found only in groups of 1-2, surrounded by hydrophobic residues, in the transmembrane domain. Thus, the transmembrane and extramembrane regions can be identified by the number of contiguous hydrophilic or hydrophobic amino acids in the 30 primary sequence of the receptor, in addition to the constraints on the length of the hydrophobic segments given above. The boundaries between the transmembrane and extramembrane regions are often defined by the presence of charged or polar residues at the beginning or end of a stretch of hydrophobic amino acids. The locations of the CA 02204491 1997-0~-0~
mutations in the receptors of the present invention are described on the basis of these models and can be specifically defined by the specific amino acid numbers of the residues being mllt~ted.
By these criteria, the third intracellular loop is defined as 5 the hydrophilic loop connecting the hydrophobic, putative tr~n~membrane domains V and VI. For example, in hamster ,~2 adrenergic receptor, the third intracellular loop would refer to amino acids 221 through 273. In accordance with the principles described above, the beginning of this loop is defined by the presence of Arg22 10 (a charged residue at the end of the hydrophobic stretch of residues 198-220) and Lys273 (a charged residue at the beginning of the hydrophobic stretch of residues 274-298). In the human NPY1 receptor (PCT International Application Publication Nos. W093/09227 published 13 May 1993 and WO93/24515 published 9 December 1993, the 15 contents of both of which are hereby incorporated by reference), the third intracellular loop refers to amino acids #233-260 (Figure 3). In accordance with the principles described above, the beginning of this loop is defined by the presence of Lys233 (a charged residue at the end of the long stretch of hydrophobic residues comprising helix 5) and 20 Arg260 (a charged residue at the beginning of the long stretch of hydrophobic residues comprising helix 6). In the rat NPY1 receptor, the third intracellular loop refers to amino acids #232-259 (Eva, C., et al., FEBS Lett. 271:81, 1990). In accordance with the principles described above, the be~inning of this loop is defined by the presence of 25 Lys232 (a charged residue at the end of the long stretch of hydrophobic residues comprising helix 5) and Arg259 (a charged residue at the beginning of the long stretch of hydrophobic residues comprising helix 6). In the human and rat NPY2 receptors, the third intracellular loop refers to amino acids #241-268 (Gerald, C., et al., PCT International 30 Application Publication No. WO95/21245, the contents of which are hereby incorporated by reference). In accordance with the principles described above, the be~innin~ of this loop is defined by the presence of Arg241 (a charged residue at the end of the long stretch of hydrophobic residues comprising helix 5) and Lys268 (a charged residue at the CA 02204491 1997-o~-o~
WO 96/14331 PCT/US95tl4377 beginning of the long stretch of hydrophobic residues comprising helix 6). In the human NPY4 receptor, the third intracellular loop refers to amino acids #236-263 (Bard, J.A., et al., PCT International Application Publication No. WO95/17906, the contents of which are hereby 5 incorporated by reference). In accordance with the principles described above, the beginning of this loop is defined by the presence of Arg236 (a charged residue at the end of the long stretch of hydrophobic residue.s comprising helix 5) and Gln263 (a polar residue at the beginning of the long stretch of hydrophobic residues comprising helix 6). In the rat 10 NPY4 receptor, the third intracellular loop refers to amino acids #236-263 (Bard, J.A., et al., PCT International Application Publication No.
WO95/17906). In accordance with the principles described above, the beginning of this loop is defined by the presence of Arg236 (a charged residue at the end of the long stretch of hydrophobic residues 15 comprising helix 5) and Arg263 (a charged residue at the beginning of the long stretch of hydrophobic residues comprising helix 6).
The present invention pertains to modified neuropeptide Y receptors having deletions, replacements or additions in the third intracellular domain. Methods of designing and making modified 20 receptors are provided. The modified receptors are uncoupled from or are poorly coupled to their respective neuropeptides. However, these modif1ed receptors bind agonists with high affinity in the absence of G protein coupling. Because of their high intrinsic affinity for agonists, these modified receptors may be used in high 25 throughput binding assays to identify compounds that bind to the receptor with high affinity, regardless of whether these compounds are agonists or antagonists. The invention includes the DNA
encoding the modif1ed receptors, the modified receptors, assays employing the modified receptors, cells expressing the modified 30 receptors, substances identified through the use of the modified receptors including specific modulators of the modified receptors, and the use of these substances in treating diseases, including obesity, diabetes, cardiovascular, and neurological disorders. Modulators identified in this process are useful as therapeutic agents.
CA 02204491 1997-0~-05 Modulators, as described herein, include but are not limited to agonists, antagonists, suppressors and inducers.
Modified receptors may include genetic variants, both natural and induced. Induced modified receptors may be derived by a S variety of methods, including but not limited to, site-directed mutagenesis. Techniques for nucleic acid and protein manipulation are well-known in the art and are described generally in Methods in Enzymology and in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989).
It is known that there is a substantial amount of redundancy in the various codons which code for specific amino acids. Therefore, this invention is also directed to those DNA
sequences which contain alternative codons which code for the eventual translation of the identical amino acid. For purposes of this specification, a sequence bearing one or more replaced codons will be defined as a degenerate variation. Also included within the scope of this invention are mutations either in the DNA sequence or the translated protein which do not subst~nti~lly alter the ultimate physical properties of the expressed protein. For example, substitution of valine for leucine, arginine for Iysine, or asparagine for glutamine may not cause a change in functionality of the polypeptide.
It is known that DNA sequences coding for a peptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide. Methods of altering the DNA sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand.
As used herein, a "functional derivative" of a modified receptor is a compound that possesses a biological activity (either functional or structural) that is substantially .simil~r to the biological activity of the modified receptor. The term "functional derivative"
is intended to include the "fragments," "variants," "degenerate CA 02204491 1997-0~-0~
variants," "analogs" and "homologues" or to "chemical derivatives"
of modified receptors. The term "fragment" is meant to refer to any polypeptide subset of modif1ed receptors. The term "variant" is meant to refer to a molecule subst~nti~lly similar in structure and function to either the entire modified receptor molecule or to a fragment thereof. A molecule is "subst~nti~lly similar" to a modified receptor if both molecules have subst~nti~lly similar structures or if both molecules possess similar biological activity.
Therefore, if the two molecules possess subst~nti~lly similar activity, they are considered to be variants even if the structure of one of the molecules is not found in the other or even if the two amino acid sequences are not identical.
The term "analog" refers to a molecule substantially similar in function to either the entire modifed receptor molecule or to a fragment thereof.
"Substantial homology" or "substantial similarity", when referring to nucleic acids means that the segments or their complementary strands, when optimally aligned and compared, are identical with a~pro~liate nucleotide insertions or deletions, in at least 50% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize to a strand or its complement.
The nucleic acids claimed herein may be present in whole cells or in cell lysates or in a partially purified or subst~nti~lly purified form. A nucleic acid is considered substantially purified when it is purified away from environmental cont~min~nt,s. Thus, a nucleic acid sequence isolated from cells is considered to be substantially purified when purified from cellular components by standard methods while a chemically synthesized nucleic acid sequence is considered to be substantially purified when purified from its chemical precursors.
Nucleic acid compositions of this invention may be derived from genomic DNA or cDNA, prepared by synthesis or by a combination of techniques.
The natural or synthetic nucleic acids encoding the modified G-coupled protein receptors of the present invention may be CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95/14377 incorporated into expression vectors. Usually the expression vectors incorporating the modified receptors will be suitable for replication in a host. Examples of acceptable hosts include, but are not limited to, prokaryotic and eukaryotic cells.
The phrase "recombinant expression system" as used herein means a subst~nti~lly homogenous culture of suitable host org~ni~m~ that stably carry a recombinant expression vector. Examples of suitable hosts include, but are not limited to, bacteria, yeast, fungi, insect cells, plant cells and m~mm~ n cells. Generally, cells of the 10 expression system are the progeny of a single ancestral transformed cell.
The cloned modified receptor DNA obtained through the methods described herein may be recombinantly expressed by molecular cloning into an expression vector cont~inin~ a suitable 15 promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant modified receptor. Techniques for such manipulations are fully described in Sambrook, J., et al., supra, and are well known in the art.
Expression vectors are defined herein as DNA sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, bluegreen algae, plant cells, insect cells, fungal cells and 25 ~nim~l cells.
Specifically designed vectors allow the shllttling of DNA
between hosts such as bacteria-yeast or bacteria-~nim~l cells or bacteria-fungi or bacteria-invertebrate cells. An appropriately constructed expression vector should contain: an origin of replication 30 for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initi~te RNA synthesis. A strong promoter is one which causes CA 02204491 1997-0~-0~
mRNAs to be initiated at high frequency. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
A variety of m~mm~ n expression vectors may be used to express recombinant modified receptor in m~mm~ n cells.
Commercially available m~mm~ n expression vectors which may be suitable for recombinant modified receptor expression, include but are not limited to, pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC
37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and ~ZD35 (ATCC 37565), pCI-neo (Promega).
A variety of bacterial expression vectors may be used to express recombinant modified receptor in bacterial cells.
Commercially available bacterial expression vectors which may be suitable for recombinant modified receptor expression include, but are not limited to pET1 la (Novagen), lambda gtl 1 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
A variety of fungal cell expression vectors may be used to express recombinant modified receptor in fungal cells.
Commercially available fungal cell expression vectors which may be suitable for recombinant modified receptor expression include but are not limited to pYES2 (Invitrogen), Pichia expression vector (Invitrogen).
A variety of insect cell expression vectors may be used to express recombinant receptor in insect cells. Commercially available insect cell expression vectors which may be suitable for recombinant expression of modified receptor include but are not limited to pBlue Bac III (Invitrogen).
An expression vector cont~ining DNA encoding modified receptor may be used for expression of modified receptor in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such CA 02204491 1997-0~-0~
WO 96/14331 PCr/US95114377 as E. coli, fungal cells such as yeast, m~mm~ n cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Cell lines derived from m~mm~lian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC
CCL 86), CV-l (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL
92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I
(ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC
CCL 171).
The expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-cont~ining cells are clonally propagated and individually analyzed to determine whether they produce modified receptor protein. Identification of modified receptor expressing host cell clones may be done by several means, including but not limited to immunological reactivity with anti-modified receptor antibodies.
Expression of modified receptor DNA may also be performed using in vitro produced synthetic mRNA or native mRNA. Synthetic mRNA or mRNA isolated from modified receptor producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
The term "substantial homology", when referring to polypeptides, indicates that the polypeptide or protein in question exhibits at least about 30% homology with the naturally occurring protein in question, usually at least about 40% homology.
CA 02204491 1997-0~-0~
WO 96/14331 PCI'/US95114377 The modified receptors may be expressed in an appropriate host cell and used to discover compounds that affect the modified receptor. Preferably, the modified receptors are expressed in a m~mm~ n cell line, including but not limited to, COS-7, CHO or L
5 cells, or an insect cell line, including but not limited to, Sf9 and Sf21, and may be used to discover ligands that bind to the receptor and alter or stim~ te its function. The modified receptors may also be produced in bacterial, fungal or yeast expression systems.
The expression of the modified receptor may be detected 10 by use of a radiolabeled ligand specific for the receptor. For example, for the ,~2 adrenergic receptor, such a ligand may be 125I-iodocyanopindolol (125I-CYP). For the NPY receptor, such a ligand may be 125I-NPY, 125I-Peptide YY (PYY) or 125I-Pancreatic polypeptide.
The specificity of binding of compounds showing affinity for the modified receptors is shown by measuring the affinity of the compounds for cells transfected with the cloned modified receptor or for membranes from these cells. Expression of the cloned modified receptor and screening for compounds that inhibit the binding of 20 radiolabeled ligand to these cells provides a rational way for rapid selection of compounds with high affinity for the receptor. These compounds may be agonists or antagonists of the receptor. Because the modified receptor does not couple well to G proteins, the agonist activity of these compounds is best assessed by using the wild-type 25 receptor, either natively expressed in tissues or cloned and exogenously expressed.
Once the modified receptor is cloned and expressed in a m~mm~ n cell line, such as COS-7 cells or CHO cells, the recombinant modified receptor is in a well-characterized environment. The 30 membranes from the recombinant cells expressing the modified receptor are then isolated according to methods known in the art. The isolated membranes may be used in a variety of membrane-based receptor binding assays. Because the modified receptor has a high affinity for agonists, ligands (either agonists or antagonists) may be CA 02204491 1997-0~-0~
identified by standard radioligand binding assays. These assays will measure the intrinsic affinity of the ligand for the receptor.
The present invention provides methods of generating modified NPY receptors. Such methods generally comprise the deletion 5 of at least one nucleotide from the third intracellular domain of the receptor. Additional methods include, but are not limited to, enzymatic or chemical removal of amino acids from the third intracellular domain of the receptor. One method of generating modified NPY receptors comprlses:
(a) isolating DNA encoding an NPY receptor;
(b) altering the DNA of step (a) by deleting at least one nucleotide from DNA encoding the third intracellular domain of the NPY receptor or disrupting the amphipathic helix at the N- or C-terminus of the third intracellular domain by replacement with 15 nucleotides or addition of nucleotides coding for non-helical protein sequence;
(c) isolating the altered DNA;
(d) expressing the altered DNA; and (e) recovering the modified NPY receptor.
20 The third intracellular domain of a G-protein coupled receptor is located between the fifth and sixth hydrophobic transmembrane domains of the receptor.
The present invention provides methods of identifying compounds that bind to modified NPY receptors. Methods of 25 identifying compounds are exemplified by an assay, comprising:
a) cloning a neuropeptide Y receptor;
b) altering the DNA sequence encoding the third intracellular domain of the cloned receptor;
c) splicing the altered receptor into an expression 30 vector to produce a construct such that the altered receptor is operably linked to transcription and translation signals suff cient to induce expression of the receptor upon introduction of the construct into a prokaryotic or eukaryotic cell;
CA 02204491 1997-0~-0~
WO 96/14331 PCI~/US95/14377 d) introducing the construct into a prokaryotic or eukaryotic cell which does not express the altered receptor in the absence of the introduced construct; and e) incubating cells or membranes isolated from cells 5 produced in step c with a quantifiable compound known to bind to the receptors, and subsequently adding test compounds at a range of concentrations so as to compete the quantifiable compound from the receptor, such that an IC50 for the test compound is obtained as the concentration of test compound at which 50% of the quantifiable 10 compound becomes displaced from the receptor.
The present invention is also directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding modified receptors or which modulate the function of modified receptor protein. Compounds which modulate these 15 activities may be DNA,RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding modified receptor, or the function of modified receptor protein.
Compounds that modulate the expression of DNA or RNA encoding 20 modified receptor or the function of modified receptor protein may be detected by a variety of assays. The assay may be a simple "yes/no" assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression 25 or function in a standard sample.
Kits cont~inin.~ modified receptor DNA, antibodies to modified receptor, or modified receptor protein may be prepared.
Such kits are used to detect DNA which hybridizes to modif1ed receptor DNA or to detect the presence of modified receptor protein 30 or peptide fragments in a sample. Such characterization is useful for a variety of purposes including but not limited to forensic, taxonomic or epidemiological studies.
The DNA molecules, RNA molecules, recombinant protein and antibodies of the present invention may be used to screen CA 02204491 1997-0~-0~
and measure levels of modified receptor DNA, modified receptor RNA or modified receptor protein. The recombinant proteins, DNA
molecules, RNA molecules and antibodies lend themselves to the forrnnl~tion of kits suitable for the detection and typing of modified 5 receptor. Such a kit would comprise a compartmentalized carrier suitable to hold in close confinement at least one container. The carrier would further comprise reagents such as recombinant modified receptor protein or anti-modified receptor antibodies suitable for detecting modified receptor. The carrier may also 10 contain a means for detection such as labeled antigen or enzyme substrates or the like.
Ph~rm~ceutically useful compositions comprising modulators of modified receptor activity, may be form~ ted according to known methods such as by the admixture of a 15 pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Ph~rm~ceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective ~lrnini~tration~ such compositions will contain an effective amount of the protein, DNA, RNA, or 20 modulator.
Therapeutic or diagnostic compositions of the invention are ~lmini~tered to an individual in amounts sufflcient to treat or diagnose disorders. The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and 25 age. Other factors include the mode of ~lmini.~tration.
The ph~rrn~ceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
The term "chemical derivative" describes a molecule 30 that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may ~ttenll~te undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Exarnples of such CA 02204491 1997-0~-0~
moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages. Alternatively, co-5 ~lmini~tration or sequential ~dmini~tration of other agents may bedesirable.
The present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present 10 invention. The compositions containing compounds identified according to this invention as the active ingredient can be ~(lmini~tered in a wide variety of therapeutic dosage forms in conventional vehicles for ~lmini~tration. For example, the compounds can be ~lmini~tered in such oral dosage forms as tablets, 15 capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspenslons, syrups and emulsions, or by injection. Likewise, they may also be ~tlmini~tered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or 20 intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
Advantageously, compounds of the present invention may be ~lmini~tered in a single daily dose, or the total daily dosage may be ~clmini~tered in divided doses of two, three or four times 25 daily. Furthermore, compounds for the present invention can be ~clmini~tered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be ~dmini.~tered in the form of a transdermal delivery system, the 30 dosage a-lmini~tration will, of course, be continuous rather than intermittent throughout the dosage regimen.
For combination treatment with more than one active agent, where the active agents are in separate dosage formulations, -CA 02204491 1997-0~-0~
the active agents can be ~lmini~tered concurrently, or they each can be ~-lmini~tered at separately staggered times.
The dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors 5 including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of ~clmini~tration; the renal and hepatic function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective 10 amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the 15 distribution, equilibrium, and elimin~tion of a drug.
The modified G-protein coupled receptors of the present invention are exemplified herein by the neuropeptide Y receptors.
Deletion mutagenesis of the ~2-adrenergic receptor has shown that none of the hydrophobic clusters of amino acids (the putative 20 transmembrane helices) could be deleted without substantial loss of binding. In contrast, most of the connecting loops could be deleted without affecting the ligand binding properties of the receptor. This indicates that these hydrophilic loops are not required for ligand binding to the receptor, suggesting that the ligand binding pocket is 25 located predomin~ntly within the transmembrane domain of the protein (Strader, et al. FASEB J .3: 182-183 (1989)). Deletions in the connecting loops that were large enough to encompass the entire loop led to steric problems, resulting in incorrect processing of the protein (Dixon, et al. EMBO J. 6: 3269-3275 (1987)). Certain connecting loop 30 deletion mutations, however, led to loss of functional activation of adenylyl cyclase by the receptor. For example, deletion of the carboxy terminal region of the third intracellular loop attenuated the ability of the receptor to activate adenylyl cyclase, and deletion of the amino terminal portion of this loop abolished adenylyl cyclase activation CA 02204491 1997-0~-0~
(Strader, et al. J. Biol. Chem. 262: 16439-16443 (1987)). Moreover, the agonist binding isotherms for these modified receptors displayed a single affinity site, suggesting altered G protein interactions. Since these modified receptors also retain their functional activation of Na+-5 H+ exchange, which is mediated through a different G protein (Barber,et al. Mol. Pharm. 41: 1056-1060 (1992)), the deletions appear not to result in gross structural perturbations of the receptor, suggesting that the changes seen in adenylyl cyclase activation are due to alteration of a specific G protein interaction. Subsequent amino acid replacements in 10 the third intracellular loop confirmed the role of this region in G
protein interaction (Cheung, et al. Mol. Pharm. 41: 1061 - 1065 (1992)).
Modified NPY1 receptors lacking between 6 and 12 amino acids in the N termin~l portion of the third intracellular loop (connecting transmembrane helices 5 and 6) may be synthesized. The 15 bottom of transmembrane helix 5 is defined by the presence of a charged amino acid (human NPY1 Lys233, rat NPYl Lys232) at the end of a series of hydrophobic amino acids. The modified receptors include the deletion of 6-12 residues following Lys233 (human) or Lys232 (rat) (i.e., I234YIRLKRRNNMM; Seq. I.D. No. 1).
20 Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., K238, R239 or R240), or replacement of such residues with alanine or a helix-disrupting residue such as proline.
A second group of modified NPY1 receptors encompass the 25 deletion of 6-13 residues at the C termin~l end of the third intracellular loop of the receptor. The C terminus of this loop is defined by the bottom of helix 6, defined by the presence of the charged residue Arg260 (human NPY1) or Arg259 (rat NPY1) preceding a stretch of hydrophobic amino acids. The modified receptors of this group have 30 deletions of 6-13 residues preceding Arg 260 in hllm~n NPY1 (i.e., KMRDNKYRSSETK259; Seq. I.D. No. 2) and proceeding Arg259 in rat NPY1 (i.e., KIRDSKYRSSETK258; Seq. I.D. No. 4). Alternatively, this sequence could be disrupted by deletion of one or more of the CA 02204491 1997-0~-0~
WO 96/14331 PCI'/US95/14377 charged residues (ie., R249, D250, K252), or replacement of such residues with ~l~nine or a helix-disrupting residue such as proline.
Modified NPY2 receptors lacking between 6 and 12 amino acids in the N terminal portion of the third intracellular loop 5 (connecting transmembrane helices 5 and 6) may be synthesized. The bottom of transmembrane helix 5 is defined by the presence of a charged amino acid (Arg241 in rat and hl1m~n) at the end of a series of hydrophobic amino acids. The modified receptors include the deletion of 6-12 residues following Arg241 (i.e., I242WSKLKNHVSPG; Seq.
10 I.D. No. 5). Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., K244, K246 or H248), or replacement of such residues with ~l~nine or a helix-disrupting residue such as proline.
A second group of modified NPY2 receptors encompass the 15 deletion of 6-13 residues at the C terminal end of the third intracellular loop of the receptor. The C terminus of this loop is defined by the bottom of helix 6, defined by the presence of the charged residue (Lys268 in human and rat) preceding a stretch of hydrophobic amino acids. The modified receptors of this group have deletions of 6-13 20 residues preceding Lys268 in human NPY2 (i.e., ANDHYHQRRQK1~267; Seq. I.D. No. 6) and proceeding Lys268 in rat NPY2 (i.e., AASDHYHQRRHKl-r267; Seq. I.D. No. 7). Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., D257, H258, H260, R262, R263, H264, K265), or 25 replacement of such residues with ~l~nine or a helix-disrupting residue such as proline.
Modified NPY4 receptors lacking between 6 and 12 amino acids in the N terminal portion of the third intracellular loop (connecting transmembrane helices 5 and 6) may be synthesized. The 30 bottom of transmembrane helix 5 is defined by the presence of a charged amino acid (Arg236 in rat and human) at the end of a series of hydrophobic amino acids. The modified receptors include the deletion of 6-12 residues following Arg236 (i.e., I237YRRLQRQGRVF in hllm~n NPY4 (Seq. I.D. No. 8) and I237YQRLQRQRRAF in rat NPY4 CA 02204491 1997-0~-0~
W0 96/14331 ~C~IUS95/14377 (Seq. I.D. No. 9)). Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., R238, R239, R242, R245), or replacement of such residues with ~l~nine or a helix-dis~ g residue such as proline.
A second group of modified NPY4 receptors encompass the deletion of 6-13 residues at the C terrnin~l end of the third intracellular loop of the receptor. The C terminus of this loop is defined by the bottom of helix 6, defined by the presence of the charged residue (Gln263 in hllm~n and Arg263 in rat) preceding a stretch of hydrophobic amino acids. The modified receptors of this group have deletions of 6-13 residues preceding Gln263 in human NPY4 (i.e., HKGTYSLRAGHMK263; Seq. I.D. No. 10) and proceeding Arg263 in rat NPY4 (i.e., HTHTCSSRVGQMK263; Seq. I.D. No. 11).
Alternatively, this sequence could be disrupted by deletion of one or more of the charged residues (ie., H251, K252, R258, H261 ), or replacement of such residues with ~l~nine or a helix-di~ g residue such as proline.
Other modified receptors encompass the deletion of 6-13 residues at either the N or C terminal end of the third intracellular loop, or replacement of residues within this region, of other members of the family of NPY receptors. The N terminus of the third intracellular loop (connecting transmembrane helices 5 and 6) is defined by the presence of a charged or polar amino acid at the end of the fifth series of hydrophobic amino acids in the sequence of the receptor (helix 5).
The C te~nimls of this loop is located at the bottom of helix 6, defined by the presence of a charged or polar residue preceding the sixth stretch of hydrophobic amino acids.
Other modified receptors encompass the addition of 5 to 10 residues at either the N or C terminal end of the third intracellular loop of the NPY1, 2 or 4 receptors such that the amphipathic nature of these regions is disrupted.
The following examples are provided to further define the invention without, however, limiting the invention to the particulars of the examples.
CA 02204491 1997-0~-05 W O96114331 PCTrUS95/14377 Deletion of 6-13 amino acids at the N-terminal portion of the third 5 intracellular loop of the hllm~n Neuropeptide Y1 receptor Modified receptor is constructed by site-directed mutagenesis of the human neuropeptide Y1 receptor cDNA by standard molecular biological techniques.
The modified DNA sequence encodes a hllm~n neuropepide 10 Yl receptor lacking between 6 and 13 amino acid residues at the N-terminal portion of the third intracellular loop. The nucleotide sequence of the modified receptor is confirmed by DNA sequencing~
As with modified ~2 receptors, the modified NPY receptor is designed so as to dismpt the proximal portion of the third intracellular loop, 15 without affecting the adjacent fifth transmembrane helix. Thus, the charged amino acid that delineates the bottom of helix 5 (Lys233) is left intact in the modified receptor, while the six to thirteen amino acids which follow it are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, beginning 20 immediately after the charged residue at the bottom of transmembrane helix 5, for example D(234-241)NPYl receptor.
25 Deletion of 6-13 amino acids at the C-terminal portion of the third intracellular loop of the hllm~n Neuropeptide Y1 receptor Modified human NPY1 receptor, lacking 13 residues at the C-terminal portion of the third intracellular loop (D(247-259)NPYI
receptor), is prepared by standard mutagenesis procedures. The 30 nucleotide sequences of the modified receptors are confirmed by DNA
sequencing. This modified human NPY1 receptor is designed so as to disrupt the distal portion of the third intracellular loop, without affecting the adjacent sixth transmembrane helix. Thus, the charged amino acid that defines the bottom of helix 6 (Lys260) is left intact, CA 02204491 1997-0~-0~
while the nearby proximal residues are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, ending immediately before the charged residues at the bottom of helix 6.
s Expression and characterization of the altered Neuropeptide Y1 receptor COS-7 cells are transfected with the modified receptor cDNA subcloned into a eukaryotic expression vector such as the eukaryotic expression vector pcDNA I/neo (Invitrogen). Cells are harvested after incubation for about 60-72 h. Membranes cont~ining the expressed receptor protein are prepared as described (C. D. Strader et al., Proc. Natl. Acad. Sci. U.S.A. 84, 4384-4388 (1987).
Binding reactions are performed in a final volume of 250 ,ul of buffer A (50 ,uM Tris, pH 7.4 cont~ining 20 mM CaC12, 5 mM
KCI, 0.2% bovine serum albumin, 10 ,uM phosphoramidon, 40 ~lg/ml bacitracin and 2 ,ug/ml leupeptin). 125I-NPY or 125I-PYY (0.1 nM) is incubated with membranes for 2 hr at 25~C before filtration over GF/C
filters presoaked in 0.1% polyethyleneimine. Filters are washed with ice cold buffer A before analysis of the bound radioactivity by scintill~tion counting.
Membranes prepared from the COS-7 cells transfected with a vector cont~ining either the wild type or the modified receptor cDNA
specifically bind a radiolabeled neuropeptide Y receptor radioligand.
The modified receptor is characterized by an absence of coupling to G
proteins, an inability to mediate the activation of second messenger systems, and an increased affinity for agonists.
The modified neuropeptide Y receptor, when expressed in m~mm~ n cells, does not stimulate G protein activation in response to the agonist NPY. In contrast, when the wild type receptor is expressed in the same cell line, activity is stimulated.
CA 02204491 1997-0~-0~
~ WO 96tl4331 PCT/US95/14377 These modified receptors have increased affinity for agonists when compared to the wild type receptor. The wild type NPYl receptor can be described pharmacologically by the relative potency of peptide ligands: neuropeptide Y= peptide YY > [Leu31Pro34]NPY >
5 NPY[2-36]>> NPY[13-36], with the affinity of NPY in the range of 0.1-10 nM, and NPY[13-36] having an affinity in the ~M range. The mutant receptor binds the agonists with the same relative order of potency. The high affinity of the agonist for the modified receptor is not affected by agents that uncouple the receptor from the G protein;
10 such agents include the nonhydrolyzable GTP analog GppNHp, sodium fluoride, and the detergent digitonin. In contrast, the wild type receptor binds agonists with two affinity states: a high affinity state, indicative of binding to the receptor-G protein complex, and a low affinity state, reflecting binding to the uncoupled receptor alone. When the receptor 15 is not optimally coupled to the G protein, a binding assay using the modified receptor will detect agonists with more sensitivity than will the identical binding assay using the wild-type receptor.
Screening Assay usin~ modified Neuropeptide Y1 receptors Transfected cells expressing recombinant modified receptor may be used to identify compounds that bind to the receptor with high affinity. This may be accomplished in a variety of ways, such as by 25 incubating the test compound in a final volume of 0.25 ml of buffer A
with membranes cont~ining 5-7 pM of the modified neuropeptide Y
receptor and 100 pM 125I-PYY or 125I-NPY for 2 hour at 25~. The reaction is stopped by filtration over GF/C glass fiber filters presoaked in 0.1% polyethyleneimine, washing with 3 x 5 ml of cold buffer A, and 30 counting the filters in a gamma counter to measure bound radioactivity.
This assay will detect a compound that has a high intrinsic affinity for the receptor. Such compounds may be either agonists or antagonists.
CA 02204491 1997-0~-0~
Deletion of 6-13 amino acids at the N-terrnin~l portion of the third intracellular loop of Neuropeptide Y receptor subtypes Modified NPY receptor subtypes (e.g., NPY2, NPY4) having deletions at the N terminal region of the third intracellular loop are constructed by site-directed mutagenesis of the neuropeptide Y
receptor cDNA by standard molecular biological techniques.
The modified DNA sequence encodes a neuropepide Y
receptor lacking between 6 and 13 amino acid residues at the N-terminal portion of the third intracellular loop. The nucleotide sequence of the modified receptor is confirmed by DNA sequencing. The modified NPY receptor is designed so as to disrupt the proximal portion of the third intracellular loop, without affecting the adjacent fifth transmembrane helix. Thus, the charged amino acid that delineates the bottom of helix 5 is left intact in the modified receptor, while the six to thirteen amino acids which follow it are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, be~innin~ imrnediately after the charged residue at the bottom of transmembrane helix 5.
Deletion of 6-13 amino acids at the C-terminal portion of the third intracellular loop of the Neuropeptide Y receptor Modified NPY receptor subtypes (e.g., NPY2, NPY4) having deletions at the C termin~l region of the third intracellular loop are constructed by site-directed mutagenesis of the neuropeptide Y
receptor cDNA by standard molecular biological techniques. The nucleotide sequences of the modified receptors are confirmed by DNA
sequencing. These modified NPY receptors have disruptions in the distal portion of the third intracellular loop, without affecting the adjacent sixth transmembrane helix. Thus, the polar amino acid that defines the bottom of helix 6 is left intact, while the nearby proximal CA 02204491 1997-0~-0~
WO 96/14331 PCI'IUS95/14377 residues are deleted. The size of the deletion in the present invention may vary from six to 13 amino acids in this region, ending immediately before the polar residues at the bottom of helix 6.
Expression and characterization of modified NPY Receptor The modified receptor is subcloned into an expression vector such as pRC/CMV (Invitrogen,San Diego, CA) and expressed in 10 m~mm~ n cells bytransfection. Approximately 72hours after transfection, cells are harvested for radioligand binding assays.
For binding assays, the membranes are prepared by harvesting the cells in ice-cold lysis buffer (5 mg Tris, pH 7.4; 2 mM
EDTA), followed by 15 min centrifugation at 38,000 x g. The 15 membrane pellet is then resuspended in buffer A. Equilibrium binding to the wild type or modified NPY receptor is performed in a final volume of 0.25 ml cont~ining membranes, 100 pM 125I-PYY, and serial dilution of the competing ligands. Binding reactions are incubated for 2 hr at 25~C, and terminated by rapid filtration over 20 GF/C filters pre-soaked in 0.1% polyethyleneimine. The radioactivity is quantified with a Packard gamma counter.
These modified receptors have increased affinity for agonists when compared to the wild type receptor. The wild type "atypical NPY1" or NPY4 receptor that mediates feeding behavior can 25 be described phar~nacologically by the high affinity of neuropeptide Y, peptide YY, NPY[2-36], and [Leu31Pro34]NPY, and the lower affinity of more truncated analogs NPY[13-36] and NPY [20-36], and structurally by its sequence homology (>45% at the DNA level) to the NPY1 receptor. The affinity of NPY for the atypical Y1 receptor 30 subtype is in the range of 0.01-10 nM, and that for NPY[13-36] is in the 0.1-10 ~M range. The mutant receptor binds the agonists with the same relative order of potency as the wild type receptor. The high affinity of the agonist for the modified receptor is not affected by agents that uncouple the receptor from the G protein; such agents include the 35 nonhydrolyzable GTP analog GppNHp, sodium fluoride, and the CA 02204491 1997-0~-05 detergent digitonin. In contrast, the wild type receptor binds agonists with two affinity states: a high affinity state, indicative of binding to the receptor-G protein complex, and a low affinity state, reflecting binding to the uncoupled receptor alone. When the receptor is not optimally S coupled to the G protein, a binding assay using the modified receptor will detect agonists with more sensitivity than will the identical binding assay using the wild-type receptor. Other NPY receptor subtypes (NPY2, NPY3, and others) are also defined pharmacologically by the relative potencies of peptide ligands for these receptors and str~cturally 10 by their sequence similarity to the NPY 1 receptor. Mutant receptors having deletions in the third intracellular loop have similar orders of potency as the corresponding wild type receptor, but with higher affinity than the wild type receptor in the absence of G protein coupling.
These modified NPY receptors are readily used in a screening assay to detect compounds that bind with high affinity to the NPY receptor subtype, regardless of whether these compounds are agonists or antagonists.
Cloning and Expression of Modified NPY Receptor cDNA into Bacterial Expression Vectors Recombinant modified receptor is produced in a bacterial expression system such as E. coli. The modified receptor expression 25 cassette is transferred into an E. coli expression vector; expression vectors include but are not limited to, the pET series (Novagen). The pET vectors place modified receptor expression under control of the tightly regulated bacteriophage T7 promoter. Following transfer of this construct into an E. coli host which contains a chromosomal copy of the 30 T7 RNA polymerase gene driven by the inducible lac promoter, expression of modified receptor is induced by addition of an approl,liate lac substrate (IPTG) is added to the culture. The levels of expressed modified receptor are determined by the assays described herein.
CA 02204491 1997-0~-0~
Cloning and Expression of Modified NPY Receptor cDNA into a Vector for Expression in Insect Cells Baculovirus vectors derived from the genome of the AcNPV virus are designed to provide high level expression of cDNA in the Sf9 line of insect cells (ATCC CRL# 1711). Recombinant baculovirus expressing modified receptor cDNA is produced by the following standard methods (InVitrogen Maxbac Manual): the modified receptor cDNA constructs are ligated into the polyhedrin gene in a variety of baculovirus transfer vectors, including the pAC360 and the BlueBac vector (InVitrogen). Recombinant baculoviruses are generated by homologous recombination following co-transfection of the baculovirus transfer vector and linearized AcNPV genomic DNA [Kitts, P.A., Nuc. Acid. Res. 18, 5667 (1990)] into Sf9 cells. Recombinant pAC360 viruses are identified by the absence of inclusion bodies in infected cells and recombinant pBlueBac viruses are identified on the basis of ~-galactosidase expression (Summers, M. D. and Smith, G. E., Texas Agriculture Exp. Station Bulletin No. 1555). Following plaque purification, modified receptor expression is measured.
Authentic modified receptor is found in association with the infected cells. Active modified receptor is extracted from infected cells by hypotonic or detergent lysis.
Alternatively, the modified receptor is expressed in the Drosophila Schneider 2 cell line by cotransfection of the Schneider 2 cells with a vector cont~ining the modified receptor DNA downstream and under control of an inducible metallothionin promoter, and a vector encoding the G418 resistant neomycin gene. Following growth in the presence of G418, resistant cells are obtained and induced to express modified receptor by the addition of CuSO4. Identification of modulators of the modified receptor is accomplished by assays using either whole cells or membrane preparations.
CA 02204491 1997-0~-0~
WO 96/14331 PCI~/US9~/14377 Cloning of Modified NPY Receptor cDNA into a yeast expression vector S Recombinant modified receptor is produced in the yeast S.
cerevisiae following the insertion of the modified receptor cDNA
cistron into expression vectors designed to direct the intracellular or extracellular expression of heterologous proteins. In the case of intracellular expression, vectors such as EmBLyex4 or the like are ligated to the modified receptor cistron [Rinas, U. et al., Biotechnology 8, 543-545 (1990); Horowitz B. et al., J. Biol. Chem. 265, 4189-4192 (1989)]. For extracellular expression, the modified receptor cistron is ligated into yeast expression vectors which fuse a secretion signal. The levels of expressed modified receptor are determined by the assays described herein.
Purification of Recombinant Modified NPY Receptor Recombinantly produced modified receptor may be purified by a variety of procedures, including but not limited to antibody affinity chromatography.
Modified receptor antibody affinity columns are made by adding the anti-modified receptor antibodies to Affigel-10 (Biorad), a gel support which is pre-activated with N-hydroxysuccinimide esters such that the antibodies form covalent linkages with the agarose gel bead support. The antibodies are then coupled to the gel via amide bonds with the spacer arm. Thè rem~ining activated esters are then quenched with 1 M ethanolamine HCI (pH 8). The column is washed with water followed by 0.23 M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) together with appropriate membrane solubilizing agents such as detergents, and the cell culture supernatants or cell extracts cont~ining solubilized modified receptor or modified CA 02204491 1997-0~-0~
WO 96/14331 PCI/US9Stl4377 receptor subunits are slowly passed through the column. The column is then washed with phosphate-buffered saline (PBS) supplemented with detergents until the optical density (A280) falls to background; then the protein is eluted with 0.23 M glycine-HCI (pH 2.6) supplemented with S detergents. The purified modified receptor protein is then dialyzed against PBS.
10 Cloning and Expression of Modified NPY Receptor in M~mm~ n Cell System A modified receptor is cloned into a m~mm~ n expression vector. The m~mm~ n expression vector is used to transform a m~mm~ n cell line to produce a recombinant m~mm~ n cell line.
15 The recombinant m~mm~ n cell line is cultivated under conditions that permit expression of the modified receptor. The recombinant m~mm~ n cell line or membranes isolated from the recombinant m~mm~lian cell line are used in assays to identify compounds that bind to the modified receptor.
Screening Assay Recombinant cells co-lt~ DNA encoding a modified 25 NPY receptor, membranes derived from the recombinant cells, or recombinant modified receptor preparations derived from the cells or membranes may be used to identify compounds that modulate modified NPY receptor activity. Modulation of such activity may occur at the level of DNA, RNA, protein or combinations thereof. One method of 30 identifying compounds that modulate modified NPY receptor, comprises:
(a) mixing a test compound with a solution cont~ining modified NPY receptor to form a mixture;
CA 02204491 1997-05-0~
WO 96114331 PCT/US95tl4377 (b) measuring modified NPY receptor activity in the mixture; and (c) comparing the modified NPY receptor activity of the mixture to a standard.
Formulation of Pharmaceutical Compositions Compounds identified by the method of Example 13 are 10 formulated into pharmaceutical compositions according to standard method.s. The compounds or pharmaceutical compositions are used either alone or in combination with other compounds or compositions for the treatment of ~nim~l.s (including humans) in need of treatment.
Conditions requiring treatment include but are not limited to obesity, 15 regulation of apetite, congestive heart failure, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac and cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, and Huntington's, Alzheimer's and Parkinson's dlseases.
Methods of Treatment ~nim~ (including hllm~ns) having a condition, the 25 condition being characterized by factors selected from altered levels of neuropeptide Y, altered activities of neuropeptide Y, altered levels of neuropeptide Y receptor activity, altered neuropeptide Y receptor activity, and combinations thereof, are treated with compounds or derivatives of compounds identified by the screening method or 30 pharmaceutical compositions comprising the compounds or derivative.s of compounds identified by the screening method.
~ nim~l.s (including humans) having a condition selected from obesity, diabetes, anxiety, hypertension, cocaine withdrawal, congestive heart failure, memory enhancement, cardiac vasospasm, CA 02204491 1997-0~-0~
cerebral vasospasm, pheochromocytoma and ganglioneuroblastoma, Huntington's Disease, Alzheimer's Disease, Parkinson's disease, and combinations thereof, are treated with a therapeutically effective amount of compounds or derivatives of compounds identified by the 5 screening method or pharmaceutical compositions comprising the compounds or derivatives of compounds identif1ed by the screening method.
While the foregoing specification teaches the principles of 10 the present invention, with examples provided for the purpose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptions, or modifications, as come within the scope of the following claims and its equivalents.
CA 0220449l 1997-0~-0~
W O 96/14331 PCTrUS95/14377 SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: STRADER, CATHERINE D.
CASCIERI, MARGARET A.
MACNEIL, DOUGLAS J.
(ii) TITLE OF INVENTION: MODIFIED NEUROPEPTIDE Y RECEPTORS
(iii) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: MARY A. APPOLLINA
(B) STREET: 126 EAST LINCOLN AVENUE
(C) CITY: RAHWAY
(D) STATE: NEW JERSEY
(E) COUNTRY: US
(F) ZIP: 07065-0900 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/335,017 (B) FILING DATE: 07-NOV-1994 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: APPOLLINA, MARY A.
(B) REGISTRATION NUMBER: 34,087 (C) REFERENCE/DOCKET NUMBER: 19339Y PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (908) 594-3462 (B) TELEFAX: (908) 594-4720 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide CA 0220449l l997-0~-0~
W O 96/14331 PCTrUS95/14377 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ile Tyr Ile Arg Leu Lys Arg Arg Asn Asn Met Met (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Lys Met Arg Asp Asn Lys Tyr Arg Ser Ser Glu Thr Lys (2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 411 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Met Asn Ser Thr Leu Phe Ser Gln Val Glu Asn His Ser Asp Phe Leu Val His Ser Asn Phe Ser Glu Lys Asn Ala Gln Leu Leu Ala Phe Glu Asn Asp Asp Cys His Leu Pro Leu Ala Met Ile Phe Thr Leu Ala Leu Ala Tyr Gly Ala Val Ile Ile Leu Gly Val Ser Gly Asn Leu Ala Leu Ile Ile Ile Ile Leu Lys Gln Lys Glu Met Arg Asn Val Thr Asn Ile Leu Ile Val Asn Leu Ser Phe Ser Asp Leu Leu Val Ala Ile Met Cys Leu Pro Leu Thr Phe Val Tyr Thr Leu Met Asp His Trp Val Phe Gly CA 0220449l l997-0~-0~
Glu Ala Met Cys Lys Leu Asn Pro Phe Val Gln Cys Val Ser Ile Thr Val Ser Ile Phe Ser Leu Val Leu Ile Ala Val Glu Arg His Gln Leu Ile Ile Asn Pro Arg Gly Trp Arg Pro Asn Asn Arg His Ala Tyr Val ~ly Ile Ala Val Ile Trp Val Leu Ala Val Ala Ser Ser Leu Pro Phe ~eu Ile Tyr Gln Val Met Thr Asp Glu Pro Phe Gln Asn Val Thr Leu Asp Ala Tyr Lys Asp Lys Tyr Val Cys Phe Asp Gln Phe Pro Ser Asp Ser His Arg Leu Ser Tyr Thr Thr Leu Leu Leu Val Leu Gln Tyr Phe Gly Pro Leu Cys Phe Ile Phe Ile Cys Tyr Phe Lys Ile Tyr Ile Arg ~eu Lys Arg Arg Asn Asn Met Met Asp Lys Ser Glu Gly Arg Phe His ~er Pro Asn Leu Gly Gln Val Glu Gln Asp Gly Arg Ser Gly His Gly Leu Met Arg Asp Asn Lys Tyr Arg Ser Ser Glu Thr Lys Arg Ile Asn Ile Met Leu Leu Ser Ile Val Val Ala Phe Ala Val Cys Trp Leu Pro Leu Thr Ile Phe Asn Thr Val Phe Asp Trp Asn His Gln Ile Ile Ala ~hr Cys Asn His Asn Leu Leu Phe Leu Leu Cys His Leu Thr Ala Met ~le Ser Thr Cys Val Asn Pro Ile Phe Tyr Gly Phe Leu Asn Lys Asn Phe Gln Arg Asp Leu Gln Phe Phe Phe Asn Phe Cys Asp Phe Arg Ser Arg Asp Asp Asp Tyr Glu Thr Ile Ala Met Ser Thr Met His Thr Asp Val Ser Lys Thr Ser Leu Lys Gln Ala Ser Pro Val Ala Phe Lys Lys CA 02204491 1997-0~-0~
W O 96/14331 PCTrUS95/14377 - 45 ~
Ile Asn Asn Asn Asp Asp Asn Glu Lys Ile Xaa (2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Lys Ile Arg Asp Ser Lys Tyr Arg Ser Ser Glu Thr Lys l 5 l0 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Ile Trp Ser Lys Leu Lys Asn His Val Ser Pro Gly l 5 l0 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ala Asn Asp His Tyr His Gln Arg Arg Gln Lys Thr Thr l 5 l0 CA 0220449l l997-0~-0~
W O96/14331 PCTtUS9Stl4377 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Ala Ser Asp His Tyr His Gln Arg Arg His Lys Thr Thr (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ile Tyr Arg Arg Leu Gln Arg Gln Gly Arg Val Phe (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ile Tyr Gln Arg Leu Gln Arg Gln Arg Arg Ala Phe CA 02204491 l997-0~-0~
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
His Lys Gly Thr Tyr Ser Leu Arg Ala Gly His Met Lys (2) INFORMATION FOR SEQ ID NO:ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
His Thr His Thr Cys Ser Ser Arg Val Gly Gln Met Lys
Claims (33)
1. Isolated DNA encoding a modified neuropeptide Y
receptor, the modified receptor comprising a neuropeptide Y receptor having seven transmembrane domains and the modified neuropeptide Y
receptor having deletions, replacements or additions in the third intracellular domain.
receptor, the modified receptor comprising a neuropeptide Y receptor having seven transmembrane domains and the modified neuropeptide Y
receptor having deletions, replacements or additions in the third intracellular domain.
2. The DNA of Claim 1 wherein the modified neuropeptide Y receptor is NPY4 receptor.
3. The DNA of Claim 1 wherein the modified neuropeptide Y receptor is NPY1 receptor.
4. The DNA of Claim 3 wherein the modified neuropeptide Y receptor is NPY1 receptor having deletions in the third intracellular domain.
5. Isolated RNA encoded by the isolated DNA of Claim 1 or a sequence complementary to the DNA of Claim 1.
6. The RNA of Claim 5 wherein the modified neuropeptide Y receptor is NPY4 receptor.
7. The RNA of Claim 5 wherein the modified neuropeptide Y receptor is NPY1 receptor.
8. The RNA of Claim 7 wherein the modified neuropeptide Y receptor is NPY1 receptor having deletions in the third intracellular domain.
9. An expression vector comprising the isolated DNA
of Claim 1.
of Claim 1.
10. The expression vector of Claim 9 comprising the isolated DNA which encodes NPY4 receptor.
11. The expression vector of Claim 9 comprising the isolated DNA which encodes NPY1 receptor.
12. The expression vector of Claim 11 comprising the isolated DNA which encodes NPY1 receptor having deletions in the third intracellular domain.
13. A cell comprising the expression vector of Claim 9.
14. A process for production of a modified neuropeptide Y receptor, comprising:
a) transforming a host with the isolated DNA of Claim 1 to produce a recombinant host;
b) culturing the recombinant host under conditions which allow the production of modified neuropeptide Y
receptor; and c) recovering the modified neuropeptide Y
receptor.
a) transforming a host with the isolated DNA of Claim 1 to produce a recombinant host;
b) culturing the recombinant host under conditions which allow the production of modified neuropeptide Y
receptor; and c) recovering the modified neuropeptide Y
receptor.
15. The modified neuropeptide Y receptor produced by the process of Claim 14.
16. An isolated and purified modified neuropeptide Y
receptor, the modified receptor comprising a neuropeptide Y receptor having seven transmembrane domains wherein the modified receptor has amino acids deleted from, replaced or added to the third intracellular domain.
receptor, the modified receptor comprising a neuropeptide Y receptor having seven transmembrane domains wherein the modified receptor has amino acids deleted from, replaced or added to the third intracellular domain.
17. The modified receptor of Claim 16 wherein the modified neuropeptide Y receptor is NPY4 receptor.
18. The modified receptor of Claim 16 wherein the modified neuropeptide Y receptor is NPY1 receptor.
19. The modified receptor of Claim 18 wherein the modified neuropeptide Y receptor is NPY1 receptor having deletions in the third intracellular domain.
20. A method of identifying compounds that modulate modified neuropeptide Y receptor activity, comprising:
(a) mixing a test compound with a solution containing modified neuropeptide Y receptor to form a mixture;
(b) measuring modified receptor activity in the mixture; and (c) comparing the modified receptor activity of the mixture to a standard.
(a) mixing a test compound with a solution containing modified neuropeptide Y receptor to form a mixture;
(b) measuring modified receptor activity in the mixture; and (c) comparing the modified receptor activity of the mixture to a standard.
21. The method of Claim 20 wherein the modified neuropeptide Y receptor is NPY4 receptor.
22. The method of Claim 20 wherein the modified neuropeptide Y receptor is NPY1 receptor.
23. Antibody immunologically reactive with the modified receptor of Claim 16.
24. A method of making a modified neuropeptide Y
receptor comprising:
(a) isolating DNA encoding a neuropeptide Y
receptor;
(b) altering the DNA of step (a) by deleting at least one nucleotide from DNA encoding the third intracellular domain of the NPY receptor or disrupting the amphipathic helix at the N- or C- terminus of the third intracellular domain by replacement with nucleotides or addition of nucleotides coding for non-helical protein sequence;
(c) isolating the altered DNA;
(d) expressing the altered DNA; and (e) recovering the modified receptor.
receptor comprising:
(a) isolating DNA encoding a neuropeptide Y
receptor;
(b) altering the DNA of step (a) by deleting at least one nucleotide from DNA encoding the third intracellular domain of the NPY receptor or disrupting the amphipathic helix at the N- or C- terminus of the third intracellular domain by replacement with nucleotides or addition of nucleotides coding for non-helical protein sequence;
(c) isolating the altered DNA;
(d) expressing the altered DNA; and (e) recovering the modified receptor.
25. The modified receptor of Claim 24.
26. The method of Claim 24 wherein between six and thirteen nucleotides are deleted from DNA encoding the third intracellular domain of the neuropeptide Y receptor.
27. The modified receptors of Claim 26.
28. The isolated DNA of Claim 1 wherein the modified neuropeptide Y receptor is selected from the group consisting of:
(a) D(234-241)NPY1 receptor; and (b) D(247-259)NPY1 receptor.
(a) D(234-241)NPY1 receptor; and (b) D(247-259)NPY1 receptor.
29. The isolated and purified receptor of Claim 16 wherein the modified neuropeptide Y receptor is selected from the group consisting of:
(a) D(234-241)NPY1 receptor; and (b) D(247-259)NPY1 receptor.
(a) D(234-241)NPY1 receptor; and (b) D(247-259)NPY1 receptor.
30. The isolated DNA of Claim 1 encoding a member of the family of NPY receptors having deletions, replacements or additions of the N or C terminal portions of the third intracellular loop, such that the modified receptor binds agonists in the high affinity state regardless of the presence or absence of G proteins.
31. The isolated DNA of Claim 30 encoding a member of the family of NPY receptors having deletions of the N or C terminal portions of the third intracellular loop, such that the modified receptor binds agonists in the high affinity state regardless of the presence or absence of G proteins.
32. The isolated and purified receptor of Claim 16 wherein the modified neuropeptide Y receptor is a member of a family of NPY receptors having deletions, replacements or additions of the N
or C terminal portions of the third intracellular loop, such that the modified receptor binds agonists in the high affinity state regardless of the presence or absence of G proteins.
or C terminal portions of the third intracellular loop, such that the modified receptor binds agonists in the high affinity state regardless of the presence or absence of G proteins.
33. The isolated and purified receptor of Claim 32 wherein the modified neuropeptide Y receptor is a member of a family of NPY receptors having deletions of the N or C terminal portions of the third intracellular loop, such that the modified receptor binds agonists in the high affinity state regardless of the presence or absence of G proteins.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US33501794A | 1994-11-07 | 1994-11-07 | |
| US335,017 | 1994-11-07 | ||
| PCT/US1995/014377 WO1996014331A1 (en) | 1994-11-07 | 1995-11-06 | Modified neuropeptide y receptors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2204491A1 true CA2204491A1 (en) | 1996-05-17 |
Family
ID=29405830
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2204491 Abandoned CA2204491A1 (en) | 1994-11-07 | 1995-11-06 | Modified neuropeptide y receptors |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2204491A1 (en) |
-
1995
- 1995-11-06 CA CA 2204491 patent/CA2204491A1/en not_active Abandoned
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