David L. Roman

Identification of small-molecule inhibitors of RGS4 using a high-throughput flow cytometry protein interaction assay.

Regulators of G-protein signaling (RGS) proteins are important components of signal transduction pathways initiated through G-protein-coupled receptors (GPCRs). RGS proteins accelerate the intrinsic GTPase activity of G-protein α-subunits (Gα) and thus shorten the time course and reduce the magnitude of G-protein α- and βγ-subunit signaling. Inhibiting RGS action has been proposed as a means to enhance the activity and specificity of GPCR agonist drugs, but pharmacological targeting of protein-protein interactions has typically been difficult. The aim of this project was to identify inhibitors of RGS4. Using a Luminex 96-well plate bead analyzer and a novel flow-cytometric protein interaction assay to assess Gα-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1, methyl N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/Gαo binding with 3 to 5 μM potency. It binds to RGS4, inhibits RGS4 stimulation of Gαo GTPase activity in vitro, and prevents RGS4 regulation of μ-opioid-inhibited adenylyl cyclase activity in permeabilized cells. Furthermore, CCG-4986 is selective for RGS4 and does not inhibit RGS8. Thus, we demonstrate the feasibility of targeting RGS/Gα protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins

Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((2E)-2-(1,3-benzothiazol-2-yl)-3-[9-methyl-2-(3-methylphenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]prop-2-enenitrile)], inhibit the interaction between RGS4 and Gαo with an IC50 value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 > 19 = 16 > 8 ≫ 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to Gαo. On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.

Polyplexed Flow Cytometry Protein Interaction Assay: A Novel High-Throughput Screening Paradigm for RGS Protein Inhibitors

Intracellular signaling cascades are a series of regulated protein-protein interactions that may provide a number of targets for potential drug discovery. Here, the authors examine the interaction of regulators of G-protein signaling (RGS) proteins with the G-protein Gαo, using a flow cytometry protein interaction assay (FCPIA). FCPIA accurately measures nanomolar binding constants of this protein-protein interaction and has been used in high-throughput screening. This report focuses on 5 RGS proteins (4, 6, 7, 8, and 16). To increase the content of screens, the authors assessed high-throughput screening of these RGS proteins in multiplex, by establishing binding constants of each RGS with Gαo in isolation, and then in a multiplex format with 5 RGS proteins present. To use this methodology as a higher-content multiplex protein-protein interaction screen, they established Z-factor values for RGS proteins in multiplex of 0.73 to 0.92, indicating this method is suitable for screening using FCPIA. To increase throughput, they also compressed a set of 8000 compounds by combining 4 compounds in a single assay well. Subsequent deconvolution of the compounds mixtures verified the identification of active compounds at specific RGS targets in their mixtures using the polyplexed FCPIA method. (Journal of Biomolecular Screening 2009: 610-619)

Assembly of High Order Gαq-Effector Complexes with RGS Proteins

Transmembrane signaling through Gαq-coupled receptors is linked to physiological processes such as cardiovascular development and smooth muscle function. Recent crystallographic studies have shown how Gαq interacts with two activation-dependent targets, p63RhoGEF and G protein-coupled receptor kinase 2 (GRK2). These proteins bind to the effector-binding site of Gαq in a manner that does not appear to physically overlap with the site on Gαq bound by regulator of G-protein signaling (RGS) proteins, which function as GTPase-activating proteins (GAPs). Herein we confirm the formation of RGS-Gαq-GRK2/p63RhoGEF ternary complexes using flow cytometry protein interaction and GAP assays. RGS2 and, to a lesser extent, RGS4 are negative allosteric modulators of Gαq binding to either p63RhoGEF or GRK2. Conversely, GRK2 enhances the GAP activity of RGS4 but has little effect on that of RGS2. Similar but smaller magnitude responses are induced by p63RhoGEF. The fact that GRK2 and p63RhoGEF respond similarly to these RGS proteins supports the hypothesis that GRK2 is a bona fide Gαq effector. The results also suggest that signal transduction pathways initiated by GRK2, such as the phosphorylation of G protein-coupled receptors, and by p63RhoGEF, such as the activation of gene transcription, can be regulated by RGS proteins via both allosteric and GAP mechanisms.

Allosteric Inhibition of the Regulator of G Protein Signaling–Gα Protein–Protein Interaction by CCG-4986

Regulator of G protein signaling (RGS) proteins act to temporally modulate the activity of G protein subunits after G protein-coupled receptor activation. RGS proteins exert their effect by directly binding to the activated Gα subunit of the G protein, catalyzing the accelerated hydrolysis of GTP and returning the G protein to its inactive, heterotrimeric form. In previous studies, we have sought to inhibit this GTPase-accelerating protein activity of the RGS protein by using small molecules. In this study, we investigated the mechanism of CCG-4986 [methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitro-benzenesulfinimidoate], a previously reported small-molecule RGS inhibitor. Here, we find that CCG-4986 inhibits RGS4 function through the covalent modification of two spatially distinct cysteine residues on RGS4. We confirm that modification of Cys132, located near the RGS/Gα interaction surface, modestly inhibits Gα binding and GTPase acceleration. In addition, we report that modification of Cys148, a residue located on the opposite face of RGS4, can disrupt RGS/Gα interaction through an allosteric mechanism that almost completely inhibits the Gα–RGS protein–protein interaction. These findings demonstrate three important points: 1) the modification of the Cys148 allosteric site results in significant changes to the RGS interaction surface with Gα; 2) this identifies a “hot spot” on RGS4 for binding of small molecules and triggering an allosteric change that may be significantly more effective than targeting the actual protein-protein interaction surface; and 3) because of the modification of a positional equivalent of Cys148 in RGS8 by CCG-4986, lack of inhibition indicates that RGS proteins exhibit fundamental differences in their responses to small-molecule ligands.

Mechanism of action and structural requirements of constrained peptide inhibitors of RGS proteins.

Regulators of G‐protein signaling (RGS) accelerate guanine triphosphate hydrolysis by Gα‐subunits and profoundly inhibit signaling by G protein‐coupled receptors. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We previously reported the design of a constrained peptide inhibitor of RGS4 (1: Ac‐Val‐Lys‐[Cys‐Thr‐Gly‐Ile‐Cys]‐Glu‐NH2, S‐S) based on the structure of the Gαi switch 1 region but its mechanism of action was not established. In the present study, we show that 1 inhibits RGS4 by mimicking and competing for binding with the switch 1 region of Gαi and that peptide 1 shows selectivity for RGS4 and RGS8 versus RGS7. Structure–activity relationships of analogs related to 1 are described that illustrate key features for RGS inhibition. Finally, we demonstrate activity of the methylene dithioether‐bridged peptide inhibitor, 2, to modulate muscarinic receptor‐regulated potassium currents in atrial myocytes. These data support the proposed mechanism of action of peptide RGS inhibitors, demonstrate their action in native cells, and provide a starting point for the design of RGS inhibitor drugs.

Novel Peptide Ligands of RGS4 from a Focused One-Bead, One-Compound Library

Regulators of G protein signaling accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein‐coupled receptors. The distinct expression patterns and pathophysiologic regulation of regulators of G protein signaling proteins suggest that inhibitors may have therapeutic potential. We previously reported the design, mechanistic evaluation, and structure–activity relationships of a disulfide‐containing cyclic peptide inhibitor of RGS4, YJ34 (Ac‐Val‐Lys‐c[Cys‐Thr‐Gly‐Ile‐Cys]‐Glu‐NH2, S‐S) (Roof et al., Chem Biol Drug Des, 67, 2006, 266). Using a focused one‐bead, one‐compound peptide library that contains features known to be necessary for the activity of YJ34, we now identify peptides that bind to RGS4. Six peptides showed confirmed binding to RGS4 by flow cytometry. Two analogs of peptide 2 (Gly‐Thr‐c[Cys‐Phe‐Gly‐Thr‐Cys]‐Trp‐NH2, S‐S with a free or acetylated N‐terminus) inhibited RGS4‐stimulated Gαo GTPase activity at 25–50 μm. They selectively inhibit RGS4 but not RGS7, RGS16, and RGS19. Their inhibition of RGS4 does not depend on cysteine‐modification of RGS4, as they do not lose activity when all cysteines are removed from RGS4. Peptide 2 has been modeled to fit in the same binding pocket predicted for YJ34 but in the reverse orientation.

Use of Flow Cytometric Methods to Quantify Protein‐Protein Interactions

A method is described for the quantitative analysis of protein‐protein interactions using the flow cytometry protein interaction assay (FCPIA). This method is based upon immobilizing protein on a polystyrene bead, incubating these beads with a fluorescently labeled binding partner, and assessing the sample for bead‐associated fluorescence in a flow cytometer. This method can be used to calculate protein‐protein interaction affinities or to perform competition experiments with unlabeled binding partners or small molecules. Examples described in this protocol highlight the use of this assay in the quantification of the affinity of binding partners of the regulator of G‐protein signaling protein, RGS19, in either a saturation or a competition format. An adaptation of this method that is compatible for high‐throughput screening is also provided. Curr. Protoc. Cytom. 51:13.11.1‐13.11.15.

Differential modulation of mu-opioid receptor signaling to adenylyl cyclase by regulators of G protein signaling proteins 4 or 8 and 7 in permeabilised C6 cells is Gα subtype dependent

J. Neurochem. (2009) 112, 1026–1034.

A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen

BackgroundRegulators of G protein signaling (RGSs) accelerate GTP hydrolysis by Gα subunits and profoundly inhibit signaling by G protein-coupled receptors (GPCRs). The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors may have therapeutic potential. We recently described a focused one-bead, one-compound (OBOC) library screen to identify peptide inhibitors of RGS4. Here we extend our observations to include another peptide with a different mechanism of action.ResultsPeptide 5nd (Tyr-Trp-c [Cys-Lys-Gly-Leu-Cys]-Lys-NH2, S-S) blocks the RGS4-Gαo interaction with an IC50 of 28 μM. It forms a covalent, dithiothreitol (DTT) sensitive adduct with a mass consistent with the incorporation of one peptide per RGS. Peptide 5nd activity is abolished by either changing its disulfide bridge to a methylene dithioether bridge, which cannot form disulfide bridges to the RGS, or by removing all cysteines from the RGS protein. However, no single cysteine in RGS4 is completely necessary or sufficient for 5nd activity.ConclusionThough it has some RGS selectivity, 5nd appears to be a partially random cysteine modifier. These data suggest that it inhibits RGS4 by forming disulfide bridges with the protein.