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Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput screening.

ABSTRACT: The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide) (BIIE0246). Presented here are five potent, selective, and publicly available Y2R antagonists identified by a high-throughput screening approach. These compounds belong to four chemical scaffolds that are structurally distinct from the peptidomimetic BIIE0246. In functional assays, IC(50) values between 199 and 4400 nM against the Y2R were measured, with no appreciable activity against the related NPY-Y1 receptor (Y1R). Compounds also displaced radiolabeled peptide YY from the Y2R with high affinity (K(i) values between 1.55 and 60 nM) while not displacing the same ligand from the Y1R. In contrast to BIIE0246, Schild analysis with NPY suggests that two of the five compounds behave as competitive antagonists. Profiling against a panel of 40 receptors, ion channels, and transporters found in the central nervous system showed that the five Y2R antagonists demonstrate greater selectivity than BIIE0246. Furthermore, the ability of these antagonists to penetrate the blood-brain barrier makes them better suited for pharmacological studies of Y2R function in both the brain and periphery.

SUBMITTER: Brothers SP

PROVIDER: S-EPMC2802430 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput screening.

Brothers Shaun P SP Saldanha S Adrian SA Spicer Timothy P TP Cameron Michael M Mercer Becky A BA Chase Peter P McDonald Patricia P Wahlestedt Claes C Hodder Peter S PS

Molecular pharmacology 20091016 1

The role of neuropeptide Y Y2 receptor (Y2R) in human diseases such as obesity, mood disorders, and alcoholism could be better resolved by the use of small-molecule chemical probes that are substantially different from the currently available Y2R antagonist, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide) (BIIE0246). Prese ...[more]

PMID: 19837904

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Project description:G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.

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Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput screening.

Publications

Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput screening.

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