State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides

SUMMARY The G protein-coupled receptor (GPCR) cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable receptors and enzymes with the exception of the Gα subunit, Gαs. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. We explored a chemical library of 10 12 cyclic peptides in order to expand the chemical search for inhibitors of this enzyme class. We identified two macrocyclic peptides, GN13 and GD20, that antagonize the active and inactive states of Gαs, respectively. Both macrocyclic peptides fine-tune Gαs activity with high nucleotide-binding-state selectivity and G protein class-specificity. Co-crystal structures reveal that GN13 and GD20 distinguish the conformational differences within the switch II/α3 pocket and block effector interactions. The Gαs inhibitors showed strong activity in cellular contexts through binding to crystallographically defined pockets. The discovery of cyclic peptide inhibitors targeting Gαs provides a path for further development of state-dependent GTPase inhibitors.