Rational Design of a Selective Covalent Modifier of G Protein beta gamma Subunits

G protein-coupled receptors transduce signals through heterotrimeric G protein G alpha and G beta gamma subunits, both of which interact with downstream effectors to regulate cell function. G beta gamma signaling has been implicated in the pathophysiology of several diseases, suggesting that G beta gamma 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 G beta gamma 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 G beta gamma by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies G beta Cys204, but also a second undefined site. The high potency of selenocystamine in G beta gamma inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on G beta. Using structural information about the hot spot, we developed a strategy to selectively target redox reversible compounds to a specific site on G beta gamma 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 G beta, likely G beta Cys204, and inhibits G beta gamma more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the G beta gamma hot spot through rational incorporation into molecules that noncovalently bind to G beta gamma.