Methods and reagents for discovering and using mammalian...

Details Methods and reagents for discovering and using mammalian... Methods and reagents for discovering and using mammalian...

1999-07-14

2002-11-05

Davenport, Avis M. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Cyclic peptides

C514S009100

Reexamination Certificate

active

06476187

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the cloning, expression and functional characterization of mammalian melanocortin receptor genes. The invention provides nucleic acid encoding mammalian melanocortin receptors, recombinant expression constructs comprising said nucleic acid, and mammalian cells into which said recombinant expression constructs have been introduced, and that express functional mammalian melanocortin receptors. The invention also provides a panel of such transformed mammalian cells expressing melanocortin receptors for screening compounds for receptor agonist and antagonist activity. The invention provides methods, for using such panels of melanocortin receptor-expressing mammalian cells to specifically detect and identify agonists and antagonists for each melanocortin receptor, as well as patterns of agonist and antagonist activity of said compounds for the class of melanocortin receptors. Such screening methods provide a means for identifying compounds with patterns of melanocortin agonist and antagonist activity which is associated with the capacity to influence or modify physiological function and behavior, particularly metabolism and feeding behavior.
2. Background of the Invention
The proopiomelanocortin (POMC) gene product is processed to produce a large number of biologically active peptides. Two of these peptides, &agr;-melanocyte stimulating hormone (&agr;MSH), and adrenocorticotropic hormone (ACTH) have well understood roles in control of melanocyte and adrenocortical function, respectively. Both of these hormones are also found in a variety of forms with unknown functions, for example, &ggr;-melanocyte stimulating hormone (&ggr;MSH), which has little or no ability to stimulate pigmentation (Ling et al., 1979
, Life Sci
. 25: 1773-1780; Slominski et al., 1992
, Life Sci
. 50: 1103-1108). A melanocortin receptor gene specific for each of the &agr;MSH, ACTH and &ggr;MSH hormones has been discovered by some of the present inventors (see U.S. Pat. Nos. 5,280,112, 5,532,347 and U.S. application Ser. No. 08/044,812, incorporated by reference herein). In addition, two other melanocortin receptor genes have been discovered by some of the present inventors (see Lu et al, 1994
, Nature
371: 799-802; Mountjoy et al, 1994
, Molec. Endocrinol
. 8: 1298-1308) and others (see Gantz et al., 1993
, J. Biol. Chem
. 268: 15174-15179 and Labbe et al., 1994
, Biochem
. 33: 4543-4549).
Along with the well-recognized activities of &agr;MSH in melanocytes and ACTH in adrenal and pituitary glands, the melanocortin peptides also have a diverse array of biological activities in other tissues, including the brain and immune system, and bind to specific receptors in these tissues with a distinct pharmacology (see, Hanneman et al., in
Peptide Hormone as Prohormones
, G. Martinez, ed. (Ellis Horwood Ltd.: Chichester, UK) pp. 53-82; DeWied & Jolles, 1982
, Physiol. Rev
. 62: 976-1059 for reviews). A complete understanding of these peptides and their diverse biological activities requires the isolation and characterization of their corresponding receptors. Some biochemical studies have been reported in the prior art.
Shimuze, 1985
, Yale J. Biol. Med
. 58: 561-570 discusses the physiology of melanocyte stimulating hormone.
Tatro & Reichlin, 1987
, Endocrinology
121: 1900-1907 disclose that MSH receptors are widely distributed in rodent tissues.
Sola et al., 1989
, J. Biol. Chem
. 264: 14277-14280 disclose the molecular weight characterization of mouse and human MSH receptors linked to radioactively and photoaffinity labeled MSH analogues.
Siegrist et al., 1991
, J. Receptor Res
. 11: 323-331 disclose the quantification of receptors on mouse melanoma tissue by receptor autoradiography.
Cone & Mountjoy, U.S. patent application Ser. No. 07/866,979, filed Apr. 10, 1992, disclose the isolation of human and mouse &agr;-MSH receptor genes and uses thereof (incorporated herein by reference).
Cone & Mountjoy, U.S. patent application Ser. No. 07/866,560, filed Apr. 10, 1992, disclose the isolation of human and bovine ACTH receptor genes and uses thereof (incorporated herein by reference).
Mountjoy et al., 1992
, Science
257: 1248-1251 disclose the isolation of cDNAs encoding mammalian ACTH and MSH receptor proteins.
POMC neurons are present in only two regions of the brain, the arcuate nucleus of the hypothalamus, and the nucleus of the solitary tract of the brain stem. Neurons from both sites project to a number of hypothalamic nuclei known to be important in feeding behavior, including the paraventricular nucleus, lateral hypothalamic area, and ventromedial hypothalamic nucleus. While previous reports have claimed both stimulatory and inhibitory effects of &agr;-MSH on feeding behavior (see Shimizu et al., 1989
, Life Sci
. 45: 543-552; Tsujii et al., 1989
, Brian Res. Bull
. 23: 165-169), knowledge of specific melanocortin receptors, their location within the central nervous system and the necessary pharmacological tools were not sufficiently developed at that time to allow the resolution of this issue. The present inventors have shown herein that a novel antagonist of the MC-3 and MC-4 melanocortin receptors can substantially increase food consumption in animals engaged in normal or fast-induced feeding behavior. This is consistent with expression of both MC-3 and MC-4 receptor mRNAs at these sites in in situ hybridization studies (Roselli-Rehfuss et al., 1993
, Proc. Natl. Acad. Sci. USA
90: 8856-8860; Mountjoy et al., 1994
, Molec. Endocrinol
. 8: 1298-1308). Moreover, the regulation of arcuate nucleus POMC gene expression is consistent with an inhibitory role for POMC in feeding behavior. POMC mRNA levels are decreased following a fast (Bergendahl et al., 1992
, Neuroendocrinol
. 56: 913-920; Brady et al., 1990
, Neuroendocrinol
. 52: 441-447), and a significant diurnal variation in POMC mRNA levels in the arcuate nucleus is seen in rat, with the nadir occurring around the onset of nighttime feeding at 1800 hrs (Steiner et al., 1994
, FASEB J
. 8: 479-488).
Thus, the experimental evidence indicates that POMC neurons play an important role in tonic inhibition of feeding behavior, wherein obesity results from a chronic disruption of this inhibitory tone by antagonism of central melanocortin receptors in at least one animal model (agouti).
These results reveal for the first time a need in the art for a means for characterizing mammalian melanocortin receptor agonists and antagonists in vitro for the development of compounds that affect feeding behavior in animals.
SUMMARY OF THE INVENTION
The present invention provides a biological screening system for identifying and characterizing compounds that are agonists or antagonists of mammalian melanocortin receptors. The biological screening system of the invention comprises a panel of transformed mammalian cells comprising a recombinant expression construct encoding a mammalian melanocortin receptor, and expressing said receptor thereby. The invention provides such a panel of transformed mammalian cells wherein the panel comprises cells expressing each of type of mammalian melanocortin receptor. Thus, the invention also provides nucleic acid encoding mammalian melanocortin receptors, recombinant expression constructs comprising said nucleic acid, and mammalian cells into which said recombinant expression constructs have been introduced, and that express functional mammalian melanocortin receptors. Methods for using such panels of melanocortin receptor-expressing mammalian cells to specifically detect and identify agonists and antagonists for each melanocortin receptor, as well as patterns of agonist and antagonist activity of said compounds for the class of melanocortin receptors, are also provided. Such screening methods provide a means for identifying compounds with patterns of melanocortin agonist and antagonist activity which is associated with the capacity to influence or modify metabolism and behavior in an animal, particularly feeding behavior.
Thus, the invention provides in a fi

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