Developing inhibitors of the guanosine triphosphate hydrolysis accelerating activity of Regulator of G protein Signaling-14

Regulator of G protein Signaling-14 (RGS14), an intracellular inactivator of G protein-coupled receptor (GPCR) signaling, has long been considered an undruggable protein due to its shallow and relatively featureless protein– protein interaction interface. Here, we describe the successful identification and validation of a tractable chemotype that selectively inhibits the GTPase-accelerating protein (GAP) activity of RGS14. Combining structure-guided virtual screening, ligand docking across multiple available receptor conformers, and enrichment validation, we progressed from an initial first-generation active compound, Z90276197, to over 40 second-generation active analogs with improved potency. These inhibitors are predicted to engage a conserved, solvent-exposed “canyon” in the RGS14 RGS-box, which interacts with the Gα switch I region. Predicted binding poses underscored the importance of non-polar interactions and shape complementarity over polar interactions in engaging RGS14’s shallow Gα-binding canyon and revealed a recurring “ambidextrous” pattern of substituent orientations. Functional GAP inhibition was confirmed in fluorescence-based and the gold-standard radioactive GTP hydrolysis assays. Two second-generation analogs, Z55660043 and Z55627844, inhibited RGS14 GAP activity in both assays and without measurable cytotoxicity. Deep learning-based scoring of predicted docking poses further supported observed affinity gains from methyl-ester additions. One analog demonstrated favorable in vivo pharmacokinetics and CNS penetration. Collectively, our findings establish an example of tractable small molecule inhibition of a regulatory interface of a G protein and illustrate how machine learning-enhanced docking can guide ligand optimization for shallow protein surfaces. This work opens the door to future development of RGS14 inhibitors as potential therapeutics for central nervous system and metabolic disorders.