Identification of non-peptidic neuromedin U receptor modulators by a robust homogeneous screening assay
Abstract
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-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 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.
Keywords:
Methods: Membrane 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 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.