Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1999-01-29
2001-10-16
Kemmerer, Elizabeth (Department: 1646)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C530S324000, C530S326000, C530S328000, C530S345000
Reexamination Certificate
active
06303749
ABSTRACT:
BACKGROUND
1. Field of the Invention
This invention relates to peptide analogs of agouti polypeptide and agouti-related polypeptide that are useful in modulating feeding behavior.
2. Related Art
Obesity is now recognized as a major health problem due in part to the association of obesity with cardiovascular disease, hypertension, and type II diabetes (Stark,
Exp. Opin. Invest. Drugs,
7:859-864 [1998]). Obesity is believed to result from the interaction of several genetic and environmental factors. Several genes have recently been identified as having a role in feeding behavior. Some of these genes are leptin, carboxypeptidase, tubby, and agouti (Stark, supra).
The agouti gene was cloned in 1992 and was found to encode a 131 amino acid polypeptide (Bultman et al.,
Cell,
71:1195-1204). The human agouti polypeptide is commonly referred to as agouti signaling protein, or “ASP”. Recent research has demonstrated that ASP binds to melanocortin-1 receptor and melanocortin-4 receptor (Stark, supra). Various attempts have been made to identify the amino acid residues of ASP that are important for binding. An ASP carboxy-terminal peptide encompassing amino acids 83-131 has been generated via expression cloning and is purportedly as active as full length ASP (Willard et al.,
Biochem.,
34:12341-12346 [1995]). Several ASP amino acid variants have been prepared by expression cloning methods. For example, Kiefer et al. (
Biochem.,
37:991-997 [1998]) have prepared various Ala scan mutants, and Perry et al. (
Genetics,
144:255-264 [1996]) have prepared two deletion mutants (desArg5-Phe14 and desArg64-Lys77) as well as various Arg, Ser and Asp substitution mutants.
Agouti related polypeptide (also referred to as “AGRP”) is known to affect feeding behavior. Mice injected with AGRP peptides have been shown to increase their food uptake, resulting in obesity and diabetes (Stark, supra). Recent research suggests that AGRP is purportedly an antagonist of melanocortin-3 receptor and melanocortin-4 receptor (Fong et al.,
Biochim. Biophys. Res. Comm.,
237:629-631 [1997]; Ollmann et al.,
Science,
278:135-138 [1997]). These melanocortin receptors have been implicated in weight regulation (Ollmann et al., supra).
The gene encoding human AGRP has been cloned and sequenced (Shutter et al.,
Genes Dev.,
11:593-602 [1997]). The corresponding human polypeptide is 132 amino acids in length, and is about 25 percent identical to human agouti polypeptide. Human AGRP contains 11 cysteines, the majority of which are located at the carboxy terminal end of the polypeptide, and form 5 disulfide bridges (Bures et al.,
Biochemistry,
37:12172-12177 [1998]).
In an effort to identify the active region of AGRP polypeptide, various peptides of the full length molecule have been prepared and tested for activity. PCT patent application WO 97/43412 (published Nov. 20, 1997) describes an AGRP peptide of amino acids 79-132. Rossi et al. (
Endocrinology,
139:4428-4431 [1998]) describe production of the AGRP peptide 83-132. Quillan et al. (
FEBS Lett.,
428:59-62 [1998]) describe production of the AGRP peptide 83-132, AGRP peptide 25-51, and AGRP peptide 54-82 using solid phase synthesis methods. Bures et al., supra, describe several AGRP peptides prepared by proteolytic digestion of full length recombinant AGRP including AGRP 102-112, 70-89, 90-92, 97-106, 105-112, 106-112, 75-91, 96-97, 75-91, 70-74, 64-67, 96-101, and 98-101.
In view of the need to better understand the biology of obesity, there is a need to identify and develop novel agonist and/or antagonist ligands of the melanocortin-3 and melanocortin-4 receptor subtypes with increased differential selectivity as compared with AGRP and ASP.
Accordingly, it is an object of this invention to provide molecules that can modulate, either positively or negatively, the biological activity of AGRP and ASP. This and other objects will be readily apparent to one of ordinary skill in the art.
SUMMARY OF THE INVENTION
In one aspect, the present invention provides a peptide selected from the group consisting of:
(a) the peptide of any of SEQ ID NOs:1-26;
(b) a peptide encoded by a nucleic acid molecule wherein the complement of the nucleic acid molecule hybridizes to any of the nucleic acid molecules of SEQ ID NOs: 27-52 under conditions of high stringency; and
(c) a peptide containing one or more conservative amino acid substitutions as compared to any of the peptides of SEQ ID NOs:1-26.
Optionally, such peptides may be acylated at the amino terminus, and an acetyl group may be used for acylation.
In another embodiment, the present invention provides an isolated nucleic acid molecule selected from the group consisting of:
(a) the nucleic acid molecule of any of SEQ ID NOs:27-52;
(b) the complement of a nucleic acid molecule that hybridizes under conditions of high stringency to any of SEQ ID NOs: 27-52; and
(c) a nucleic acid molecule encoding a peptide that contains one or more conservative amino acid substitutions as compared to the peptides of any of SEQ ID NOs: 1-26.
The invention further provides a vector comprising any of such nucleic acid molecules, as well as eukaryotic and prokaryotic host cells comprising such vectors.
DETAILED DESCRIPTION OF THE INVENTION
The section headings herein are for organizational purposes only and are not to be construed as limiting in any way the subject matter described therein.
Definitions
The term “AGRP/ASP peptides” refers to the peptides having the amino acid sequence of any of SEQ ID NOs:1-26, together with all related peptides described herein. Related peptides includes allelic variants, fragments, derivatives, substitution, deletion, and insertion variants, fusion polypeptides, and orthologs, and each amino acid of each such related peptide may be of the “D” (natural) or “L” (unnatural) configuration which corresponds to the stereochemical designation “S” and “R”, respectively, as defined in the RS system of Cahn et al. (
Pure Applied Chemistry,
45:11-30, [1974], and references therein). Such related peptides may be mature peptides, i.e., lacking a signal peptide. The AGRP/ASP peptides may or may not have amino terminal methionines, depending on the manner in which they are prepared.
As used herein, the term “AGRP/ASP peptide variants” refers to AGRP/ASP peptides whose amino acid sequences contain one or more amino acid sequence substitutions, deletions, and/or additions as compared to the AGRP/ASP peptide amino acid sequences set forth in SEQ ID NOS:1-26. Such AGRP/ASP peptide variants containing amino acids of the natural L-configuration can be prepared from the corresponding AGRP/ASP nucleic acid molecule variants, which have a DNA sequence that varies accordingly from the DNA sequences encoding the wild type AGRP/ASP peptides as set forth in SEQ ID NOS:1-26. Alternatively, such variants containing amino acids of the D-configuration (unnatural form) can be prepared synthetically using standard methods described herein (see also
Biochem. J.,
219:345-373 [1984]).
As used herein, the term “AGRP/ASP peptide derivatives” refers to AGRP/ASP peptides, variants, or fragments thereof, that have been chemically modified, as for example, by addition of one or more water soluble polymers, N-linked or O-linked carbohydrates, sugars, phosphates, and/or other such molecules, where the molecule or molecules are not naturally attached to wild-type AGRP/ASP peptides. Derivatives further includes deletion of one or more chemical groups naturally attached to the AGRP/ASP peptide.
As used herein, the terms “biologically active AGRP/ASP peptides”, “biologically active AGRP/ASP peptide fragments”, “biologically active AGRP/ASP peptide variants”, and “biologically active AGRP/ASP peptide derivatives” refer to AGRP/ASP peptides which bind to both the human melanocortin-3 receptor and to the human melanocortin-4 receptor, and have a binding affinity to one of these receptors when competed against I-
125
AGRP of no greater than
Amgen Inc.
Kemmerer Elizabeth
Levy Ron K.
Odre Steven M.
Oleski Nancy A.
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