Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2001-05-04
2003-11-11
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S071100, C435S071200, C435S471000, C435S325000, C435S252300, C435S254110, C435S320100, C536S023500, C530S350000
Reexamination Certificate
active
06645738
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to rhesus monkey (
Macaca mulatta
) DNA molecules encoding the melanocortin-5 receptor protein belonging to the rhodopsin sub-family of G-protein coupled receptors, recombinant vectors comprising DNA molecules encoding rhesus MC-5R, recombinant host cells which contain a recombinant vector encoding rhesus MC-5R, the rhesus MC-5R protein encoded by the DNA molecule, and methods of identifying selective agonists and antagonists of rhesus MC-5R.
BACKGROUND OF THE INVENTION
Melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR's). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as &agr;-, &bgr;, or &ggr;-melanocyte stimulating hormones (&agr;-, &bgr;-, &ggr;-MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
U.S. Pat. No.5,532,347, issued on Jul. 2, 1996, to Cone and Mountjoy discloses human and mouse DNA molecules which encode MC-1R (also known in the art as &agr;-MSH-R). The expressed human protein contains 317 amino acids.
U.S. Pat. No. 5,280,112 (issued Jan. 18, 1994) and U.S. Pat. No.5,554,729 (issued Sep. 10, 1996), both to Cone and Mountjoy, disclose human and mouse DNA molecules which encode MC-2R (also known in the art as ACTH-R). The human MC-2R protein contains 297 amino acids.
Mountjoy, et al. (1992
, Science
257: 1248-1251) describe DNA molecules and the concomitant protein for human MC-1R and human MC-2R.
Chhajlani, et al. (1992
, FEBS Letters
309: 417-420) also disclose a human DNA molecule comprising an open reading frame which encodes human MC1-R.
Roselli-Rehfuss, et al, (1993
, Proc. Natl. Acad. Sci
90: 8856-8860) disclose a cDNA clone encoding rat MC-3R cDNA.
U.S. Pat. No. 5,622,860 (issued Apr. 22, 1997) and U.S. Pat. No. 5,703,220 (issued Dec. 30, 1997) to Yamada and Gantz, disclose DNA molecules which encode human MC-3R and human MC-4R, respectively (see also Gantz, et al., 1993
, J. Biol. Chem
. 268(11): 8246-8250).
A DNA molecule encoding human MC-5R was also disclosed by Mountjoy, et al. (1994
, Mol. Endocrin
. 8: 1298-1308).
Chhajlani, et al. (1993
, Biochem. Biophys. Res. Comm
., 195(2): 866-873) disclose a DNA molecule which the authors state encodes MC-5R. This clone was initially designated MC2.
Fathi, et al. (1995
, Neurochenrical Research
20(1):107-113) also disclose a DNA molecule thought to encode human MC-5R. There are several sequence discrepancies when compared to the DNA molecule disclosed by Chhajlani, et al., id.
Griffon, et al. (1994
, Biochem. Biophys. Res. Comm
., 200(2): 1007-1014) disclose DNA clones from human and rat which encode MC-5R. The human DNA sequence agrees with the human DNA sequence disclosed in Fathi et al. id.
Gantz, et al. (1994
, Biochem. Biophys. Res. Comm
., 200(3): 11214-11220; see also U.S. Pat. No. 5,710,265, issued Jan. 20, 1998 to Yamada and Gantz) and Labbe, et al. (1994
, Biochemistry
33: 4543-4549) disclose DNA clones from mouse which encode MC-5R.
Barrett, et al. (1994
, J. Mol. Endocrin
. 12: 203-213) disclose DNA clones from sheep which encode MC-5R.
In rodents, MC-4R has been implicated as a key regulator of feeding behavior which regulates body weight through studies with peptide agonists and antagonists (Fan et al., 1997
, Nature
385: 165-168) and with a MC-4R knock-out mouse (Huszar et al., 1997
, Cell
88: 131-141).
Compounds that bind to such receptors were previously identified by binding to human and/or rodent receptors and evaluated for their efficacy in rodents. However, the neuroendocrine process can differ between rodents and man. It is also expected that some compounds exhibit different binding affinities for different species homologues of the same receptor (Fong et al., 1992
, J. Biol. Chem
. 267:25666-25671; Hartig et al., 1992, TIPS 13:152-159).
Before compounds can be selected as a drug candidate it is first evaluated for a physiological effect in rodents and then in the rhesus primate. It is often that one compound may be effective in one animal species but not in another. Previously, it has been impossible to determine if the failure was due to an altered melanocortin pathway in different species, or due to a compound's having a lower affinity for one particular species. Past protocols required the use of a rhesus brain membrane to determine the in vitro biochemical activity of compounds, if such protocol could be successfully employed.
It is desirable to correlate in vivo data with in vitro biochemical activity of compounds.
It is also desirable to first select compounds that are active for the rhesus receptor in vitro.
It is also desirable to identify compounds which can determine the relevance of receptor targets in rhesus and allow selection of novel drugs to treat obesity.
It is further desirable to discover new drugs which effect pathophysiological processes by modulating the effects in rhesus to identify melanocortin active process in primates, followed by human clinical trials.
The present invention addresses and meets these needs by disclosing an isolated nucleic acid fragment which expresses a form of rhesus MC-5R, recombinant vectors which house this nucleic acid fragment, recombinant host cells which expresses rhesus MC-5R and/or a biologically active equivalent, and pharmacological properties of this rhesus MC-5R protein.
SUMMARY OF THE INVENTION
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes a novel rhesus monkey (
Macaca mulatta
) melanocortin-5 receptor (rhMC-5R). The nucleic acid molecules of the present invention are substantially free from other nucleic acids.
The present invention relates to an isolated nucleic acid molecule (polynucleotide) which encodes mRNA which expresses a novel rhesus MC-5R, this DNA molecule comprising the nucleotide sequence disclosed herein as SEQ ID NO:1.
The present invention also relates to biologically active fragments or mutants of SEQ ID NO:1 which encodes mRNA expressing a novel rhesus MC-5R. Any such biologically active fragment and/or mutant will encode either a protein or protein fragment which at least substantially mimics the pharmacological properties of a wild-type MC-5R protein, including but not limited to the rhesus MC-5R receptor truncations and carboxy-terminal truncations such that these mutations encode mRNA which express a protein or protein fragment of diagnostic, therapeutic or prophylactic use and would be useful for screening for agonists and/or antagonists for MC-5R function.
A preferred aspect of this portion of the present invention is disclosed in
FIG. 1
, a rhesus cDNA molecule encoding a novel MC-5R (SEQ ID NO:1).
The isolated nucleic acid molecules of the present invention may include a deoxyribonucleic acid molecule (DNA), such as genomic DNA and complementary DNA (cDNA), which may be single (coding or noncoding strand) or double stranded, as well as synthetic DNA, such as a synthesized, single stranded polynucleotide. The isolated nucleic acid molecule of the present invention may also include a ribonucleic acid molecule (RNA).
The present invention also relates to recombinant vectors and recombinant hosts, both prokaryotic and eukaryotic, which contain the substantially purified nucleic acid molecules disclosed throughout this specification.
The present invention also relates to subcellular membrane fractions of the recombinant host cells (both prokaryotic and eukaryotic as well as both stably and transiently transformed cells) which contain the proteins encoded by the nucleic acids of the present invention. These subcellular membrane fractions will comprise either wild-type or mutant forms of rhesus melanocortin-5 receptor proteins at levels substantially above endogenous levels and hence will be useful in various assays described throughout this specification.
The present invention also relates to a substantially purified form of the rhesus melanocortin-5 receptor protein,
Fong Tung M.
Huang Ruey-Ruey C.
Van Der Ploeg Leonardus H. T.
Finnegan Alysia A.
Giesser Joanne M.
Mertz Prema
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