The high throughput screening of neuropeptide FF2 receptor ligands from Korean herbal plant extracts.
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 GTPgammaS 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 microM, and PC 315 with K(i) of 2.17 microM 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.
Seoul National University, School of Agricultural Biotechnology, San 56-1 Sillim-dong, Gwanak-gu, Seoul 151-742, Republic of Korea. eudo@snu.ac.kr
Ernest U
EU
Do
Long Zhu
LZ
Piao
Gyu
G
Choi
Young Bong
YB
Choi
Tong Mook
TM
Kang
Jaekyoon
J
Shin
Yung-Jin
YJ
Chang
Hee-Young
HY
Nam
Ho-Jin
HJ
Kim
Su-Il
SI
Kim
D020650:Combinatorial Chemistry Techniques; D034242:Cyclooctanes / Q000494:pharmacology; D004149:Dioxoles / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005454:Fluorescence Polarization / Q000379:methods*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007723:Korea; D017705:Lignans / Q000494:pharmacology; D008518:Medicine, East Asian Traditional; D019151:Peptide Library; D010936:Plant Extracts / Q000737:chemistry*; D018013:Receptors, Neuropeptide / Q000819:agonists / Q000378:metabolism*; D032141:Schisandra / Q000737:chemistry
D034242:Cyclooctanes; D004149:Dioxoles; D017705:Lignans; C511786:PC 314; D019151:Peptide Library; D010936:Plant Extracts; D018013:Receptors, Neuropeptide; C083929:neuropeptide FF receptor; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C034557:schizandrer A