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Glucagon-like peptide-1 (GLP-1) receptor is an ideal target in the development of incretin-based therapies for diabetes and obesity. Two approaches have been adopted: GLP-1 receptor agonists that mimic the effects of native GLP-1 and dipeptidyl peptidase-4 inhibitors that increase endogenous GLP-1 levels. During the past two decades, search for orally active, non-peptidic GLP-1 receptor agonists has been the focal point of research and development activities in many multinational pharmaceutical companies. Such efforts have not resulted in any success thus far. Serendipitous discovery of substituted cyclobutanes represented by Boc5 as a new class of GLP-1 receptor agonists led us to believe that a small molecule approach to class B G-protein coupled receptor agonism is no longer a fantasy but a reality. However, major obstacles still pose great challenges, and the reasons of which are discussed in this perspectives. [ABSTRACT FROM AUTHOR]

Aim: To characterize the in vitro bioactivities of rhodanine derivatives as novel peroxisome proliferator-activated receptor (PPAR) γ modulators, based on a hit (SH00012671) identified during high-throughput screening (HTS) of a diverse synthetic compound library, and to preliminarily elucidate the structure-activity relationship of this class of PPARγ agonists. Methods: Full-length PPARγ and retinoid X receptor α (RXRα), biotinylated PPAR response element (PPRE), [3H]BRL49653 (rosiglitazone), and streptavidin-coated FlashPlate or microbeads were used to measure the receptor-binding properties of various compounds based on the scintillation proximity assay (SPA) technology. A recombinant PPRE vector was transiently cotransfected with PPARγ and RXRα plasmids into the African green monkey kidney (CV-1) cells, and the effects of BRL49653 and test compounds on transcription mediated by PPARγ were determined by examining luciferase (reporter) responses. 3T3-L1 cells were employed to determine whether the compounds facilitated adipogenesis upon PPARγ activation. Results: Of the 16 000 samples screened with the SPA method, only 1 compound (SH00012671) displayed a similar binding affinity ( Ki=186.7 nmol/L) to PPARγ as BRL49653, but it was inactive in the cell-based assays. A series of rhodanine derivatives were synthesized based on the core structure of SH00012671 and 8 of them showed agonist activities in both cotransfection and pre-adipocyte differentiation assays. To reduce intrinsic cytotoxicities, the sulphur on the rhodanine was changed to oxygen. This alteration led to a decrease in receptor-binding affinities while modified analogues generally maintained agonist efficacies in the cell-based assays. Of the analogues studied, compound 31 exhibited about 70% the efficacy exerted by BRL49653 in both cotransfection and pre-adipocyte differentiation assays. Conclusion: Through minor chemical modifications on the core structure of the initial HTS hit, SH00012671 was transformed to possess both molecular (PPARγ binding) and cellular (adipogenesis) activities. The rhodanine derivatives reported here may represent a new scaffold in further understanding the molecular mechanism of agonism at PPARγ. [ABSTRACT FROM AUTHOR]

Aim: To develop homogeneous calcium mobilization assay for high-throughput screening (HTS) of mas-related gene (Mrg) receptor agonists. Methods: CHO-K1 cells stably expressing the full-length MrgD receptor and a calcium-sensitive dye were used to develop an HTS assay based on intracellular calcium influx. This method was applied to large-scale screening of a library containing 8000 synthetic compounds and natural product extracts. cAMP measurements were carried out to verify the bioactivities of the hits found by the calcium mobilization assay. Similar approaches were also employed in the identification of the MrgA1 receptor agonists following HTS of 16 000 samples. Results: EC50 values of the positive control compounds (β-alanine for MrgD receptor and dynorphin A for MrgA1 receptor) determined by the calcium mobilization assay were consistent with those reported in the literature, and the Z′ factors were 0.65 and 0.50 for MrgD and MrgA1 receptor assay, respectively. About 31 compounds for the MrgD receptor and 48 compounds for the MrgA1 receptor showing ≥20% of the maximal agonist activities found in the controls were initially identified as hits. Secondary screening confirmed that 2 compounds for each receptor possessed specific agonist activities. Intracellular cAMP level measurements indicated that the 2 confirmed hits displayed the functionality of the MrgD receptor agonists. Conclusion: A series of validation studies demonstrated that the homogeneous calcium mobilization assay developed was highly efficient, amenable to automation and a robust tool to screen potential MrgD and MrgA1 receptor agonists. Its application may be expanded to other G-protein coupled receptors that mobilize calcium influx upon activation. [ABSTRACT FROM AUTHOR]