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

Ernest U. Do, Long Zhu Piao, Gyu Choi, Young Bong Choi, Tong Mook Kang, Jaekyoon Shin, Yung Jin Chang, Hee Young Nam, Ho Jin Kim, Su il Kim

Research output: Contribution to journalArticlepeer-review

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 Ki of 1.42 μM, and PC 315 with Ki 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.

Original languageEnglish (US)
Pages (from-to)997-1004
Number of pages8
JournalPeptides
Volume27
Issue number5
DOIs
StatePublished - May 2006
Externally publishedYes

Keywords

  • CRE luciferase reporter assay
  • Fluorescence polarization
  • High throughput screening
  • Neuropeptide FF
  • Neuropeptide FF2 receptor
  • Rank order of potency

ASJC Scopus subject areas

  • Biochemistry
  • Physiology
  • Endocrinology
  • Cellular and Molecular Neuroscience

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