The high throughput screening of neuropeptide FF2 receptor ligands from Korean herbal plant extracts

Abstract

We have screened 356 libraries of Korean herbal plant extracts to find potential anti-obesity drugs. We employed the recently developed fluorescence polarization high throughput screening (FP HTS) assays of human neuropeptide FF (NPFF) receptors in 384-well microtiter plates. The primary hits were cherry-picked from the libraries and further analyzed by secondary displacement curve assays, in vitro GTPγS binding assays and cell-based CRE luciferase reporter assays. Agonists of NPFF receptors showed biphasic affinity curves while the antagonist, BIBP 3226, gave a monophasic affinity curve in competitive binding assays. We isolated and characterized two agonists of human NPFF2 receptor, PC 314 with K i of 1.42 μM, and PC 315 with K i of 2.17 μM from Schizandra chinensis. PC 314 and PC 315 have been characterized as benzoylgomisin Q (M.W. 552) and gomisin G (M.W. 536). We report that PC 314 and PC 315 are the first non-peptide, natural compounds, which bind to human NPFF2 receptors with good affinity. PC 314 and PC 315 inhibit forskolin-stimulated luciferase expression when CHO cells are co-transfected with NPFF2 receptor and CRE reporter vector. They possess the pharmacological and functional profiles of full agonists. The FP HTS system provides a specific, sensitive and reproducible methodology for studying and screening NPFF receptor ligands.