Mary W. Walker

Cloning and Functional Expression of a Human Y4 Subtype Receptor for Pancreatic Polypeptide, Neuropeptide Y, and Peptide YY

The pancreatic polypeptide family includes pancreatic polypeptide (PP), neuropeptide Y (NPY), and peptide YY (PYY). Members of the PP family regulate numerous physiological processes, including appetite, gastrointestinal transit, anxiety, and blood pressure. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype has been cloned previously. We now report the cloning of an additional Y-type receptor, designated Y4, by homology screening of a human placental genomic library with transmembrane (TM) probes derived from the rat Y1 gene. The Y4 genomic clone encodes a predicted protein of 375 amino acids that is most homologous to Y1 receptors from human, rat, and mouse (42% overall; 55% in TM). I-PYY binding to transiently expressed Y4 receptors was saturable (pK = 9.89) and displaceable by human PP family derivatives: PP (pK = 10.25) PP (pK = 10.06) > PYY (pK = 9.06) [Leu,Pro]NPY (pK = 8.95) > NPY (pK = 8.68) > PP (pK = 7.13) > PP (pK = 6.46) > PP free acid (pK < 5). Human PP decreased [cAMP] and increased intracellular [Ca] in Y4-transfected LMTK cells. Y4 mRNA was detected by reverse transcriptase-polymerase chain reaction in human brain, coronary artery, and ileum, suggesting potential roles for Y4 receptors in central nervous system, cardiovascular, and gastrointestinal function.

Expression Cloning and Pharmacological Characterization of a Human Hippocampal Neuropeptide Y/Peptide YY Y2 Receptor Subtype

The pancreatic polypeptide family includes neuropeptide Y (NPY), one of the most abundant neuropeptides in the mammalian nervous system, as well as peptide YY (PYY) and pancreatic polypeptide (PP). This peptide family is involved in numerous physiological processes such as memory, pain, blood pressure, appetite, anxiety, and circadian rhythm. Of the multiple Y-type receptors proposed for PP family members, only the Y1 subtype was cloned previously. We now report the isolation of a human Y2 (hhY2) receptor cDNA by expression cloning from a human hippocampal cDNA library, using a 125I-PYY binding assay. hhY2 cDNA encodes a predicted protein of 381 amino acids with low amino acid identity to the human Y1 receptor (31% overall; 41% transmembrane). 125I-PYY binding to transiently expressed hY2 receptors was saturable (pKd = 10.17) and displaceable by human PP family members in rank order: PYY (pKi = 9.47) ∼ NPY (pKi = 9.27) PP (pKi < 6) and by peptide analogs: NPY2-36 (pKi = 8.80) ∼ NPY13-36 (pKi = 8.55) ∼ C2-NPY (pKi = 8.54) > NPY26-36 (pKi = 6.51) ∼ [Leu31,Pro34]NPY (pKi = 6.23). Human PYY decreased [cAMP] and increased intracellular [Ca2+] in hY2-transfected 293 cells.

FOOD INTAKE IN FREE-FEEDING AND ENERGY-DEPRIVED LEAN RATS IS MEDIATED BY THE NEUROPEPTIDE Y5 RECEPTOR

The new neuropeptide Y (NPY) Y5 receptor antagonist CGP 71683A displayed high affinity for the cloned rat NPY Y5 subtype, but > 1, 000-fold lower affinity for the cloned rat NPY Y1, Y2, and Y4 subtypes. In LMTK cells transfected with the human NPY Y5 receptor, CGP 71683A was without intrinsic activity and antagonized NPY-induced Ca2+ transients. CGP 71683A was given intraperitoneally (dose range 1-100 mg/kg) to a series of animal models of high hypothalamic NPY levels. In lean satiated rats CGP 71683A significantly antagonized the increase in food intake induced by intracerebroventricular injection of NPY. In 24-h fasted and streptozotocin diabetic rats CGP 71683A dose-dependently inhibited food intake. During the dark phase, CGP 71683A dose-dependently inhibited food intake in free-feeding lean rats without affecting the normal pattern of food intake or inducing taste aversion. In free-feeding lean rats, intraperitoneal administration of CGP 71683A for 28 d inhibited food intake dose-dependently with a maximum reduction observed on days 3 and 4. Despite the return of food intake to control levels, body weight and the peripheral fat mass remained significantly reduced. The data demonstrate that the NPY Y5 receptor subtype plays a role in NPY-induced food intake, but also suggest that, with chronic blockade, counterregulatory mechanisms are induced to restore appetite.

Expression Cloning of a Rat Hypothalamic Galanin Receptor Coupled to Phosphoinositide Turnover

The neuropeptide galanin is widely distributed throughout the central and peripheral nervous systems and participates in the regulation of processes such as nociception, cognition, feeding behavior, and insulin secretion. Multiple galanin receptors are predicted to underlie its physiological effects. We now report the isolation by expression cloning of a rat galanin receptor cDNA distinct from GALR1. The receptor, termed GALR2, was isolated from a rat hypothalamus cDNA library using a125I-porcine galanin (125I-pGAL) binding assay. The GALR2 cDNA encoded a protein of 372 amino acids exhibiting 38% amino acid identity with rat GALR1. Binding of125I-pGAL to transiently expressed GALR2 receptors was saturable (K D = 0.15 nm) and displaceable by galanin peptides and analogues in rank order: porcine galanin ≃ M32 ≃ M35 ≃ M40 ≥ galanin-(1–16) ≃ M15 ≃ [d-Trp2]galanin-(1–29) > C7 ≫ galanin-(3–29). This profile resembles that of the rat GALR1 receptor with the notable exception that [d-Trp2]galanin exhibited significant selectivity for GALR2 over GALR1. Activation of GALR2 receptors with porcine galanin and other galanin analogues increased inositol phospholipid turnover and intracellular calcium levels in stably transfected Chinese hamster ovary cells and generated calcium-activated chloride currents in Xenopus oocytes, suggesting that the rat GALR2 receptor is primarily coupled to the activation of phospholipase C.

Distribution of the neuropeptide Y Y2 receptor mRNA in rat central nervous system

Our group has recently reported the expression cloning of the human neuropeptide Y Y2 receptor DNA and subsequently the cloning of the rat homologue. These studies have made it possible to localize the mRNA encoding this NPY receptor subtype in rat tissues. We have, thus, carried out in situ hybridization studies, using radiolabeled oligonucleotide probes to the rat Y2 receptor mRNA, to determine the distribution of Y2 mRNA in rat brain and limited peripheral ganglia. Probe specificity was confirmed by testing antisense and sense probes in transfected cells. In rat brain, hybridization signals obtained with the antisense probes were discrete and were restricted to neuronal profiles in specific subregions of the cortex, hippocampus, amygdala, thalamus, hypothalamus, mesencephalon and pons. Among the regions exhibiting the most intense labeling were the CA3 region of the hippocampus, the arcuate nucleus of the hypothalamus and layer 3 of the piriform cortex. Other regions containing labeled neurons included the medial amygdala, the centromedial thalamic nucleus, the dorsal raphe, the dorsal motor nucleus of the vagus and the trigeminal ganglion. The present results indicate that the mRNA encoding the Y2 receptor is discretely localized in the rat brain and that the distribution is generally consistent with previous radioligand-binding studies. This study should help clarify the relationship between the Y2 receptor distribution and functional studies of NPY receptor subtype classification and provides further evidence for the involvement of the Y2 receptor in multiple physiological processes.

Design, synthesis and SAR of a series of 2-substituted 4-amino-quinazoline neuropeptide Y Y5 receptor antagonists.

The design of a novel series of NPY-Y5 receptor antagonists is described. Key elements for the design were the identification of weak Y5 hits from a Y1 program, results from a combinatorial approach and database mining. This led to the discovery of the quinazoline 4 and the aryl-sulphonamide moiety as major components of the pharmacophore for Y5 affinity. The synthesis and SAR towards CGP71683A is described.

A structure-activity analysis of the cloned rat and human Y4 receptors for pancreatic polypeptide

We cloned and expressed the rat Y4 receptor for pancreatic polypeptide (PP). Structure-activity profiles derived from 125I-PP binding assays and [cAMP] radioimmunoassays reveal a selective receptor interaction with rat PP vs. neuropeptide Y (NPY) or peptide YY (PYY). Rat and human Y4 receptor clones share 75% amino acid identity. Based on [cAMP] radioimmunoassay, the human Y4 receptor exhibits a less selective interaction with rat PP vs. NPY or PYY and a greater dependence on N-terminal PP residues, relative to rat Y4. Differences in sequence and structure-activity profiles suggest the rat be used with caution to model human Y4 receptor function.

Discovery of potent and selective small molecule NPY Y5 receptor antagonists.

The discovery of a new class of sulfonamide NPY Y5 receptor antagonists is described. Optimization of this series led to the identification of compounds with high affinity for the hY5 subtype and excellent selectivity over the other NPY receptor subtypes. The SAR for this series was examined and a model for understanding the ligand-receptor interactions was developed.

5-(2'-Pyridyl)-2-aminothiazoles: alkyl amino sulfonamides and sulfamides as potent NPY(5) antagonists.

Synthesis, SAR and physico-chemical properties of an alkyl aminothiazole series 8 and 16 are described. 2-Pyridylaminothiazole based compounds such as 8c and 16a exhibit high affinity at the NPY(5) receptor with desirable cLogPs and solubilities. However, they also suffer from high in vitro and in vivo clearance. Compound 16a partially inhibits the feeding behavior elicited by i.c.v. injection of the selective NPY(5) agonist [cPP(1-7), NPY(19-23), Ala(31), Aib(32), Gln(34)]-human pancreatic polypeptide polypeptide (cPP).

High-throughput synthesis optimization of sulfonamide NPY Y5 antagonists.

A series of sulfonamide neuropeptide Y Y5 antagonists was optimized by preparation of sets of analogues using high-throughput synthesis and purification techniques. Testing of these compounds for their ability to bind to the human NPY Y5 receptor revealed separate SAR trends for sulfonamide amides versus sulfonamide ureas versus sulfonamide amines. By understanding these SAR trends, potent compounds were identified in all three series.