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

• Published in: Acta pharmacologica Sinica Vol. 29; no. 4; pp. 517 - 527
• Main Authors: Meng, Tao, Su, Hao-ran, Binkert, Christoph, Fischli, Walter, Zhou, Ling, Shen, Jing-kang, Wang, Ming-wei
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.04.2008; Nature Publishing Group
• Subjects: Animals; Biological Assay; Biomedical and Life Sciences; Biomedicine; CHO Cells; Cricetinae; Cricetulus; Drug Evaluation, Preclinical - methods; Humans; Immunology; Internal Medicine; Ligands; Medical Microbiology; original-article; Pharmacology/Toxicology; Protein Binding; Receptors, Neurotransmitter - agonists; Receptors, Neurotransmitter - antagonists & inhibitors; Reproducibility of Results; Scintillation Counting - methods; Structure-Activity Relationship; Triticum aestivum; Vaccine; 化验方法; 生物化学; 结构-活性关系; neuromedin U; receptors; modulators; structure-activity relationship
• ISSN: 1671-4083; 1745-7254
• DOI: 10.1111/j.1745-7254.2008.00769.x

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.