Abstract

G protein-coupled receptors transduce signals through heterotrimeric G protein Gα and Gβγ subunits, both of which interact with downstream effectors to regulate cell function. Gβγ signaling has been implicated in the pathophysiology of several diseases, suggesting that Gβγ could be an important pharmaceutical target. Previously, we used a combination of virtual and manual screening to find small molecules that bind to a protein-protein interaction “hot spot” on Gβγ and block regulation of physiological effectors. One of the most potent and effective compounds from this screen was selenocystamine. In this study, we investigated the mechanism of action of selenocystamine and found that selenocysteamine forms a covalent complex with Gβγ by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies GβCys204, but also a second undefined site. The high potency of selenocystamine in Gβγ inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on Gβ. Using structural information about the “hot spot,” we developed a strategy to selectively target redox reversible compounds to a specific site on Gβγ using peptide carriers such as SIGCAFKILGY(-cysteamine) [SIGC(-cysteamine)]. Mass spectrometry and site-directed mutagenesis indicate that SIGC(-cysteamine) specifically and efficiently leads to cysteamine (half-cystamine) modification of a single site on Gβ, likely GβCys204, and inhibits Gβγ more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the Gβγ “hot spot” through rational incorporation into molecules that noncovalently bind to Gβγ.