Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-11-25
2001-11-13
Kunz, Gary L. (Department: 1647)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S070100, C435S071100
Reexamination Certificate
active
06316203
ABSTRACT:
BACKGROUND OF THE INVENTION
Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the sequence listing and the claims.
Neuropeptide Y (NPY) is a member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY and its relatives (peptide YY or PYY, and pancreatic polypeptide or PP) elicit a broad range of physiological effects through activation of at least five G protein-coupled receptor subtypes known as Y1, Y2, Y3, Y4 (or PP), and the “atypical Y1”. The role of NPY as the most powerful stimulant of feeding behavior yet described is thought to occur primarily through activation of the hypothalamic “atypical Y1” receptor. This receptor is unique in that its classification was based solely on feeding behavior data, rather than radioligand binding data, unlike the Y1, Y2, Y3, and Y4 (or PP) receptors, each of which were described previously in both radioligand binding and functional assays. Applicants now report the use of a
125
I-PYY-based expression cloning technique to isolate a rat hypothalamic cDNA encoding an “atypical Y1” receptor referred to herein as the Y5 subtype. Applicants also report the isolation and characterization of a Y5 homolog from human hippocampus. Protein sequence analysis reveals that the Y5 receptor belongs to the G protein-coupled receptor superfamily. Both the human and rat homolog display≦42% identity in transmembrane domains with the previously cloned “Y-type” receptors. Rat brain localization studies using in situ hybridization techniques verified the existence of Y5 receptor mRNA in rat hypothalamus. Pharmacological evaluation revealed the following similarities between the Y5 and the “atypical Y1” receptor. 1) Peptides bound to the Y5 receptor with a rank order of potency identical to that described for the feeding response: NPY≧NPY
2-36
=PYY=[Leu
31
, Pro
34
]NPY>>NPY
13-36
. 2) The Y5 receptor was negatively coupled to cAMP accumulation, as had been proposed for the “atypical Y1” receptor. 3) Peptides activated the Y5 receptor with a rank order of potency identical to that described for the feeding response. 4) The reported feeding “modulator” [D-Trp
32
]NPY bound selectively to the Y5 receptor and subsequently activated the receptor. 5) Both the Y5 and the “atypical Y1” receptors were sensitive to deletions or modifications in the midregion of NPY and related peptide ligands. These data support the identity of the Y5 receptor as the previously described “atypical Y1”, and furthermore indicate a role for the Y5 receptor as a potential target in the treatment of obesity, metabolism, and appetite disorders.
The peptide neurotransmitter neuropeptide Y (NPY) is a 36 amino acid member of the pancreatic polypeptide family with widespread distribution throughout the mammalian nervous system. NPY is considered to be the most powerful stimulant of feeding behavior yet described (Clark et al., 1984; Levine and Morley, 1984; Stanley and Leibowitz, 1984). Direct injection into the hypothalamus of satiated rats, for example, can increase food intake up to 10-fold over a 4-hour period (Stanley et al., 1992). The role of NPY in normal and abnormal eating behavior, and the ability to interfere with NPY-dependent pathways as a means to appetite and weight control, are areas of great interest in pharmacological and pharmaceutical research (Sahu and Kalra, 1993; Dryden et al., 1994). Any credible means of studying or controlling NPY-dependent feeding behavior, however, must necessarily be highly specific as NPY can act through at least 5 pharmacologically defined receptor subtypes to elicit a wide variety of physiological functions (Dumont et al., 1992). It is therefore vital that knowledge of the molecular biology and structural diversity of the individual receptor subtypes be understood as part of a rational drug design approach to develop subtype selective compounds. A brief review of NPY receptor pharmacology is summarized below and also in Table 1.
Table 1: Pharmacologically Defined Receptors for NPY and Related Pancreatic Polypeptides
Rank orders of affinity for key peptides (NPY, PYY, PP, [Leu
31
, Pro
34
]NPY, NPY
2-36
, and NPY
13-36
) are based on previously reported binding and functional data (Schwartz et al., 1990; Wahlestedt et al., 1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). Data for the Y2 receptor were disclosed in U.S. Pat. No. 5,545,549 filed on Feb. 3 1994, the foregoing contents of which are hereby incorporated by reference. Data for the Y4 receptor were disclosed in U.S. Pat. No. 5,516,653 filed on Dec. 28 1993, the contents of which are hereby incorporated by reference. Missing peptides in the series reflect a lack of published information.
TABLE 1
Affinity (pK
i
or pEC
50
)
Receptor
11 to 10
10 to 9
9 to 8
8 to 7
7 to 6
<6
Y1
NPY
NPY
2-36
NPY
13-36
PP
PYY
[Leu
31
,
Pro
34
]
NPY
Y2
PYY
NPY
13-36
[Leu
31
,
NPY
Pro
34
]
NPY
2-
NPY
36
PP
Y3
NPY
[Pro
34
]
NPY
13-36
PP
PYY
NPY
Y4
PP
PYY
NPY
NPY
13-36
[Leu
31
,
NPY
2-36
Pro
34
]
NPY
atypical
PYY
NPY
13-36
Y1
NPY
(feeding)
NPY
2-
36
[Leu
31
,
Pro
34
]
NPY
NPY Receptor Pharmacology
NPY receptor pharmacology has historically been based on structure/activity relationships within the pancreatic polypeptide family. The entire family includes the namesake pancreatic polypeptide (PP), synthesized primarily by endocrine cells in the pancreas; peptide YY (PYY), synthesized primarily by endocrine cells in the gut; and NPY, synthesized primarily in neurons (Michel, 1991; Dumont et al., 1992; Wahlestedt and Reis, 1993). All pancreatic polypeptide family members share a compact structure involving a “PP-fold” and a conserved C-terminal hexapeptide ending in Tyr
36
(or Y
36
in the single letter code). The striking conservation of Y
36
has prompted the reference to the pancreatic polypeptides' receptors as “Y-type” receptors (Wahlestedt et al., 1987), all of which are proposed to function as seven transmembrane-spanning G protein-coupled receptors (Dumont et al., 1992).
The Y1 receptor recognizes NPY≧PYY>>PP (Grundemar et al., 1992). The receptor requires both the N- and the C-terminal regions of the peptides for optimal recognition. Exchange of Gln
34
in NPY or PYY with the analogous residue from PP (Pro
34
), however, is well-tolerated. The Y1 receptor has been cloned from a variety of species including human, rat and mouse (Larhammar et al, 1992; Herzog et al, 1992; Eva et al, 1990; Eva et al, 1992). The Y2 receptor recognizes PYY~NPY>>PP and is relatively tolerant of N-terminal deletion (Grundemar et al., 1992). The receptor has a strict requirement for structure in the C-terminus (Arg
33
-Gln
34
-Arg
35
-Tyr
36
-NH
2
); exchange of Gln
34
with Pro
34
, as in PP, is not well tolerated. The Y2 receptor has recently been cloned (disclosed in U.S. Pat. No. 5,545,549, filed Feb. 3, 1994). The Y3 receptor is characterized by a strong preference for NPY over PYY and PP (Wahlestedt et al., 1991). [Pro
34
]NPY is reasonably well tolerated even though PP, which also contains Pro
34
, does not bind well to the Y3 receptor. The Y3 receptor (Y3) has not yet been cloned. The Y4 receptor (disclosed in U.S. Pat. No. 5,516,653, filed Dec. 28, 1993) binds PP>PYY>NPY. Like the Y1, the Y4 requires both the N- and the C-terminal regions of the peptides for optimal recognition (U.S. Pat. No 5,516,653). The “atypical Y1” or “feeding” receptor was defined exclusively by injection of several pancreatic polypeptide analogs into the paraventricular nucleus of the rat hypothalamus which stimulated feeding behavior with the following rank order: NPY
2-36
≧NPY~PYY~[Leu
31
,Pro
34
]NPY>NPY
Branchek Theresa
Gerald Christophe P. G.
Walker Mary W.
Weinshank Richard L.
Cooper & Dunham LLP
Gucker Stephen
Kunz Gary L.
Synaptic Pharmaceutical Corporation
White John P.
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