Endogenous Intracellular Metabolites Allosterically Modulate GPCR-Gα Interface

Exogenous allosteric modulators of GPCRs have been extensively investigated. To date, a few endogenous intracellular allosteric modulators are known with inconclusive binding information and their associated phenotypes. This limited understanding stems from the non-availability of robust computational techniques facilitating automated cavity identification, its topology-specific ligand design and synthesis. Here, we introduce Gcoupler, which leverages an integrative approach combining de novo ligand design, statistical methods, and Graph Neural Networks for rationally predicting high-affinity ligands for allosteric cavities, including the GPCR-Gα interface. We tested Gcoupler in decrypting the cellular metabolites that could intracellularly modulate the Ste2p-mediated pheromone-induced programmed cell death in yeast. Our rigorous interrogation using Gcoupler and experimental approaches, including genetic screening, multiomics, metabolite-binding residue missense mutants, and functional assays, identified endogenous hydrophobic metabolites (e.g. sterols), as direct intracellular allosteric modulators of Ste2p. Molecular simulations further indicate metabolites binding to GPCR-Gα obstruct downstream signaling, possibly via cohesive effect. Finally, by utilizing isoproterenol-induced, GPCR-mediated human and neonatal rat cardiac hypertrophy models, we observed elevated metabolite levels attenuate hypertrophic response, reinforcing the evolutionary relevance of this mechanism.