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

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 assessments and structure-activity relationship （sAR）） analyses. Methods： Membrane preparations of Chinese hamster ovary cells （CHO-K1） stably expressing hNMU-R 1, [^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 subsequent 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-R1 measured by the SPA method was consistent with that reported in the literature, and the Z＇ factor for this HTS assay was 0.81. 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-R1-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-expressing cells, but none displayed antagonist activity. Conclusion： The homogeneous hNMU-R1 binding assay is an efficient and robust tool for screening potential hNMU-R modulators. Two non-selective hNMU-R agonists discovered are of low molecular weight nature with novel chemical structures. The preliminary SAR investigation suggests that both the triphenyl and guanidinol groups are crucial to the bioactivities observed.