Rational Design of a Selective Covalent Modifier of G Protein βγ Subunits

Abstract

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

The Expanding Roles of GβγSubunits in G Protein–Coupled Receptor Signaling and Drug Action

“…Similar results were obtained in a mouse model that already had established heart failure for a persistent period of time, suggesting that these inhibitors could be beneficial for both the prevention and management of heart failure (Casey et al, 2010). The use and development of Gbg inhibitors has been reviewed, but it remains to be seen if inhibitors for specific combinations might be developed (Smrcka, 2008;Dessal et al, 2011).…”

Understanding Molecular Recognition by G protein βγ Subunits on the Path to Pharmacological Targeting

“…The G␤␥ inhibitory compounds could be divided into two general classes on the basis of binding mechanism. One class, which included M119 (NSC119910; 2-(3,4,5-trihydroxy-6-oxoxanthen-9-yl)cyclohexane-1-carboxylic acid) and the highly related molecule gallein (3Ј,4Ј,5Ј,6Ј-tetrahydroxyspiro[2-benzofuran-3,9Ј-xanthene]-1-one) , referred to together as M119/ gallein, bound via a reversible noncovalent mechanism (Seneviratne et al, 2011), whereas another class, represented by selenocystamine, formed redox-reversible covalent adducts with G␤␥ (Dessal et al, 2011). Many of these redox-dependent compounds targeted a cysteine residue (Cys204) in the G␤ hot spot to form reversible mixed disulfides.…”

Direct-reversible binding of small molecules to G protein βγ subunits

“…Another possibility is promiscuous-aggregation based binding of small molecule aggregates to protein surfaces leading to a general non-specific inhibition of protein function [6]. In the course of this work we found two mechanisms for inhibition by two general groups of compounds; 1) reversible covalent reactions with cysteine residues in the protein interaction surface ([8] and Dessal and Smrcka unpublished data), and in the work described here, 2) reversible non-covalent binding of compounds to Gβγ. Understanding these mechanisms is critical to determining whether use of these compounds can be leads for a viable therapeutic strategy.…”