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

Abstract

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 β-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.

Screening for positive allosteric modulators of cholecystokinin type 1 receptor potentially useful for management of obesity

“…Monoclonal cell lines (HEK-293 CCK1R, HEK-293 SNAP-CCK1R, HEK-293 CCK1R-SmBiT LgBiT-ARRB2) and membranes (HEK-293 SNAP-CCK1R) for primary and secondary assays were generated as described previously, 16 but utilizing constructs carrying the coding sequence of human CCK1R (NM_000730.3). The pcDNA3.1/Zeo(+)-CCK1R construct was transfected to develop a monoclonal HEK-293 CCK1R cell line with selection induced by Zeocin, whereas HEK-293 SNAP-CCK1R cells were selected by addition of G418 after transfection with pcDNA3.1(+) SNAP-CCK1R.…”

“…Fusion vectors for generating HEK-293 CCK1R-SmBiT LgBiT-ARRB2 cells were obtained from Promega (Promega Corp., Madison, WI, USA) and prepared as described in a previous report. 16 The monoclonal cell line was obtained via limiting dilution of Hygromycin- and Blasticidin-resistant cells. The HEK-293 CCK1R cells expressed 57,000 ± 3,000 copies of this receptor on their surface, as determined by direct CCK radioligand binding analysis.…”

“…The pcDNA3.1/Zeo(+)-CCK1R construct was transfected to develop a monoclonal HEK-293 CCK1R cell line with selection induced by Zeocin, whereas HEK-293 SNAP-CCK1R cells were selected by addition of G418 after transfection with pcDNA3.1(+) SNAP-CCK1R. Fusion vectors for generating HEK-293 CCK1R-SmBiT LgBiT-ARRB2 cells were obtained from Promega (Promega Corp., Madison, WI, USA) and prepared as described in a previous report 16 . The monoclonal cell line was obtained via limiting dilution of Hygromycin-and Blasticidin-resistant cells.…”

“…Tag-lite SNAP-Lumi-4-Terbium (Cisbio US, Inc., Bedford, MA, USA, #SSNPTBD, TR-FRET donor) were prepared as described previously 16 . HF488-CCK-9s, a CCK peptide where the free amino-group of sulfated (Thr²⁸ ,Nle³¹)-Cholecystokinin (25-33) had been ligated with HiLyte Fluor 488 (HF488), was utilized as the orthosteric ligand carrying the TR-FRET acceptor fluorophore.…”

“…multiplex assay: Cisbio Gs dynamic cAMP assays (Cisbio US, Inc., Bedford, MA, USA) and β-Arrestin-2 recruitment assays were developed and performed as reported previously 16,17 , with the following modifications to enable multiplex analysis of both signaling pathways: The monoclonal cell line HEK-293 CCK1R-SmBiT LgBiT-ARRB2 was maintained in culture and was harvested at 80-90% confluency at passage numbers below 35. After centrifugation at 300 x g for 3 min, the cell pellet was re-suspended in assay buffer (HBSS with Mg 2+ and Ca 2+ (Hank's Balanced Salt Solution, Gibco, #24020117, 5 mM HEPES (hydroxyethyl piperazineethanesulfonic acid), 0.1% BSA) containing NanoBiT substrate and diluted to target cell density.…”