Establishment of robust functional assays for the characterization of neuropeptide Y (NPY) receptors: identification of 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile as selective NPY type 5 receptor antagonist

The human Neuropeptide Y (NPY) receptors 1 (hY 1), 2 (hY 2), 4 (hY 4), and the mouse type 5 (mY 5) receptor were expressed in human embryonic kidney 293 (HEK293) cells. The receptors bound a radioiodinated NPY ligand with high affinity and various NPY analogs competed for binding in a receptor selective-manner. Similarly, cAMP-inhibition and GTPγS binding assays were established. The four NPY receptors were further tested in the fluorimetric imaging plate reader (FLIPR) format, a cellular high-throughput assay, in the absence and presence of chimeric G proteins, Gq o5, Gq i5 and Gq i9. The receptors stimulated transient calcium release only in the presence of chimeric G proteins. While hY 1, hY 2 and hY 4 receptors coupled to Gq o5, Gq i5 and Gq i9, the mY 5 receptor stimulated transient calcium release only when co-expressed with Gq i9. Using an in silico screening approach we identified a small molecule 3-(5-benzoyl-thiazol-2-ylamino)-benzonitrile (compound 1), which bound to the mY 5 receptor with high affinity ( K i = 32.1 ± 1.8 nM), competitively antagonized NPY-mediated GTPγS binding and calcium stimulation with high potency, and had no affinity for other NPY receptors. These data show that NPY receptors can be functionally coupled to the FLIPR readout, allowing for high throughput compound testing and identification of novel molecules.