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

Abstract

Heterotrimeric guanine nucleotide-binding proteins (G proteins) composed of three subunits α, β, γ mediate activation of multiple intracellular signaling cascades initiated by G protein-coupled receptors (GPCRs). Previously our laboratory identified small molecules that bind to Gβγ and interfere with or enhance binding of select effectors with Gβγ. To understand the molecular mechanisms of selectivity and assess binding of compounds to Gβγ, we used biophysical and biochemical approaches to directly monitor small molecule binding to Gβγ. Surface plasmon resonance (SPR) analysis indicated that multiple compounds bound directly to Gβγ with affinities in the high nanomolar to low micromolar range but with surprisingly slow on and off rate kinetics. While the koff was slow for most of the compounds in physiological buffers, they could be removed from Gβγ with mild chaotropic salts or mildly dissociating collision energy in a mass-spectrometer indicating that compound-Gβγ interactions were non-covalent. Finally, at concentrations used to observe maximal biological effects the stoichiometry of binding was 1:1. The results from this study show that small molecule modulation of Gβγ-effector interactions is by specific direct non-covalent and reversible binding of small molecules to Gβγ. This is highly relevant to development of Gβγ targeting as a therapeutic approach since reversible, direct binding is a prerequisite for drug development and important for specificity.

Intrinsic plasticity induced by group II metabotropic glutamate receptors via enhancement of high-threshold KV currents in sound localizing neurons

“…GPCRs can modulate intrinsic currents via the G α subunit or G βγ complex (review in Hille, 1994; Dascal, 2001). Application of the cell permeable G βγ blocker gallein (5 μM) to brain slices for 30 mins is sufficient to block G βγ complex activation (Lehmann et al, 2008; Belkouch et al, 2011; Seneviratne et al, 2011). Therefore, we incubated slices in gallein (5 μM) for >30 mins prior to recording K V currents.…”

Pharmacological profile of phosphatidylinositol 3‐kinases and related phosphatidylinositols mediating endothelinA receptor‐operated native TRPC channels in rabbit coronary artery myocytes

“…We therefore examined if the established pathway of G‐protein βγ subunits coupled to PI3K is responsible for linking ET A receptor to channel activation (Stephens et al ., 1994; Clapham and Neer, 1997; Leopolt et al ., 1998). ET A receptor stimulation of TRPC1/C5/C6 and TRPC3/C7 channel activities was inhibited by co‐application of 20 µM gallein, a G‐protein βγ subunit inhibitor (Seneviatne et al ., 2011), by over 90% in cell‐attached patches (Figure 8). In control experiments, gallein did not inhibit TRPC1/C5/C6 and TRPC3/C7 channel activities induced by 10 µM diC8‐PI(3)P in inside‐out patches (Figure 8B and C).…”

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.…”