Development of a Testing Funnel for Identification of Small-Molecule Modulators Targeting Secretin Receptors.

The secretin receptor (SCTR), a prototypical class B G protein-coupled receptor (GPCR), exerts its effects mainly by activating Gαs proteins upon binding of its endogenous peptide ligand secretin. SCTRs can be found in a variety of tissues and organs across species, including the pancreas, stomach, liver, heart, lung, colon, kidney, and brain. Beyond that, modulation of SCTR-mediated signaling has therapeutic potential for the treatment of multiple diseases, such as heart failure, obesity, and diabetes. However, no ligands other than secretin and its peptide analogs have been described to regulate SCTRs, probably due to inherent challenges in family B GPCR drug discovery. Here we report creation of a testing funnel that allowed targeted detection of SCTR small-molecule activators. Pursuing the strategy to identify positive allosteric modulators (PAMs), we established a unique primary screening assay employing a mixture of three orthosteric stimulators that was compared in a screening campaign testing 12,000 small-molecule compounds. Beyond that, we developed a comprehensive set of secondary assays, such as a radiolabel-free target engagement assay and a NanoBiT (NanoLuc Binary Technology)-based approach to detect ß-arrestin-2 recruitment, all feasible in a high-throughput environment as well as capable of profiling ligands and hits regarding their effect on binding and receptor function. This combination of methods enabled the discovery of five promising scaffolds, four of which have been validated and further characterized with respect to their allosteric activities. We propose that our results may serve as starting points for developing the first in vivo active small molecules targeting SCTRs.

Discovery of small molecule positive allosteric modulators of the secretin receptor.

The secretin receptor (SCTR) is a prototypic Class B1 G protein-coupled receptor (GPCR) that represents a key target for the development of therapeutics for the treatment of cardiovascular, gastrointestinal, and metabolic disorders. However, no non-peptidic molecules targeting this receptor have yet been disclosed. Using a high-throughput screening campaign directed at SCTR to identify small molecule modulators, we have identified three structurally related scaffolds positively modulating SCTRs. Here we outline a comprehensive study comprising a structure-activity series based on commercially available analogs of the three hit scaffold sets A (2-sulfonyl pyrimidines), B (2-mercapto pyrimidines) and C (2-amino pyrimidines), which revealed determinants of activity, cooperativity and specificity. Structural optimization of original hits resulted in analog B2, which substantially enhances signaling of truncated secretin peptides and prolongs residence time of labeled secretin up to 13-fold in a dose-dependent manner. Furthermore, we found that investigated compounds display structural similarity to positive allosteric modulators (PAMs) active at the glucagon-like peptide-1 receptor (GLP-1R), and we were able to confirm cross-recognition of that receptor by a subset of analogs. Studies using SCTR and GLP-1R mutants revealed that scaffold A, but not B and C, likely acts via two distinct mechanisms, one of which constitutes covalent modification of Cys-347GLP-1R known from GLP-1R-selective modulators. The scaffolds identified in this study might not only serve as novel pharmacologic tools to decipher SCTR- or GLP-1R-specific signaling pathways, but also as structural leads to elucidate allosteric binding sites facilitating the future development of orally available therapeutic approaches targeting these receptors.