State-selective modulation of heterotrimeric Gαs signaling with macrocyclic peptides

The G protein-coupled receptor cascade leading to production of the second messenger cAMP is replete with pharmacologically targetable proteins, 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 1012 cyclic peptides 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. Cell-permeable analogs of GN13 and GD20 modulate Gαs/Gβγ signaling in cells 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. [Display omitted] •Discovery of nucleotide-state-selective cyclic peptide binders for Gαs•Co-crystal structures reveal G protein class specificity of the cyclic peptides•Inhibition of the Gαs ON-state reduces isoproterenol-stimulated cAMP production•Sequestering the Gαs OFF-state prolongs Gβγ activation upon receptor activation Cell-permeable Gαs inhibitors regulate G protein signal transduction with high nucleotide-binding-state selectivity and class specificity.