,Identification of non-peptidic neuromedin U receptor modulators by a robust homogeneous screening a

• 作 者: Tao MENG Hao-ran SU Christoph BINKERT Walter FISCHLI Ling ZHOU Jing-kang SHEN Ming-wei WANG
• 机 构: The State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of
• 出 处: 中国药理学报（英文版）
• 发表日期: 2008年4期
• 关键词: neuromedin U receptors modulators structure-activity relationship

Aim:To develop a homogeneous binding assay for high-throughput screening (HTS) of hit compounds at human neuromedin U receptor (hNMU-R) 1 and to identify non-peptidic small molecule hNMU-R modulators through functional as-sessments and structure-activity relationship (SAR) analyses. Methods:Mem-brane preparations of Chinese hamster ovary cells (CHO-K1) stably expressing hNMU-R1, [125I]hNMU-25, and wheat germ agglutinin-coupled microbeads were used to develop an HTS assay based on scintillation proximity assay (SPA) technology. This method was applied to a large-scale screening campaign against a diverse library of 36 000 synthetic compounds or natural products and subse-quent confirmation studies. CHO-K1 cells stably expressing full-length hNMU-R1 or hNMU-R2 and a calcium-sensitive dye were employed to functionally measure intracellular calcium mobilization upon ligand stimulation. Preliminary SAR was determined based on limited structural modifications. Results:The Ki value (0.7 nmol/L) of hNMU-25 (the natural ligand) at hNMU-R 1 measured by the SPA method was consistent with that reported in the literature, and the Z’ factor for this HTS assay was 0.8 1. A total of 100 hits, showing more than 30% competitive inhibition on [125I]hNMU-25 binding to hNMU-R1, were identified initially, 3 of which were confirmed thereafter to have reasonable hNMU-Rl-binding affinities and similar chemical structures. Based on their common molecular skeleton, 203 analogs were synthesized and tested. Among the 16 analogs that retained variable hNMU-R1-binding abilities, 2 elicited calcium influx in both hNMU-R1 and hNMU-R2-ex-pressing cells, but none displayed antagonist activity. Conclusion:The homoge-neous hNMU-R1 binding assay is an efficient and robust tool for screening po-tential hNMU-R modulators. Two non-selective hNMU-R agonists discovered are of low molecular weight nature with novel chemical structures. The prelimi-nary SAR investigation suggests that both the triphenyl and guanidinol groups are crucial to the bioactivities observed.