Receptor cavity-based screening reveals potential allosteric modulators of gonadotropin receptors in carp (Cyprinus carpio)

The gonadotropins follicle stimulating hormone (FSH) and luteinizing hormone (LH) are key regulators of sexual development and the reproductive cycle in vertebrates. Unlike most G protein-coupled receptors (GPCR), the follicle-stimulating hormone receptors (FSHRs) and luteinizing hormone receptors (LHRs) have large extracellular domains containing multiple leucine-rich repeats, leading to an elaborate receptor activation mechanism via orthosteric sites that are difficult to manipulate synthetically. We used carp as a model organism to identify allosteric sites capable of receptor activation on the transmembrane domain which are spatially separated from orthosteric sites, to bypass the orthosteric mechanism, in this study. We have further generated pharmacophore hypothesis based on these cavities’ structural motifs and exposed residues. Using available online small compound libraries consisting of > 70,000 small molecules, we have therefore used a receptor cavity-based hypothesis and other screening stages to identify potential modulators of the allosteric binding site on the carp FSHR and LHR in-silico . Using in-vitro transactivation assays, we then examined the effect of four candidate compounds on FSHR and LHR compared to the activity of native ligands. Our results revealed both specific and dual effective allosteric modulators for FSHR and LHR, which can work independently or in synergy with the native ligands in a variable manner, demonstrating the potential of our approach for efficient pharmacophore-based screening.