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

Roof, Rebecca A.; Roman, David L.; Clements, Samuel T; Sobczyk-Kojiro, Katarzyna; Blazer, Levi L.; Ota, Sho; Mosberg, Henry I.; Neubig, Richard R.

doi:10.1186/1471-2210-9-9

Cited by 21 publications

(20 citation statements)

References 11 publications

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“…A great portion of the total inhibition by CCG-4986 is caused by modification at Cys148, in an allosteric site, whereas a smaller level of inhibition (approximately 30%) is caused by direct competition of CCG-4986 at Cys132 near the G␣ o binding site on RGS4. We have been able to dissect these two mechanisms by using a series of point mutants and a flow cytometry-based method that has been increasingly implemented for studying RGS proteins (Gu et al, 2007;Roof et al, 2008Roof et al, , 2009Shankaranarayanan et al, 2008;Roman et al, 2009). We recognize that CCG-4986 modifies a number of cysteine residues on RGS4; however, the lack of inhibition of RGS8 despite the presence of CCG-4986 modification demonstrates differences in the conformational changes that occur in RGS4 after binding of the inhibitor at the allosteric site marked by Cys148.…”

Section: Discussionmentioning

confidence: 99%

“…Because of their unique role in temporally regulating G protein-mediated signals, RGS proteins have emerged as an attractive drug target with considerable effort focused on developing exogenous ligands to modulate their activity (Nieuwenhuijsen et al, 2003;Young et al, 2004;Roof et al, 2006Roof et al, , 2007Roof et al, , 2008Roof et al, , 2009Roman et al, 2007b). Advantages to targeting RGS proteins include their often unique tissue distributions and the presence of accessory domains of some RGS families that can provide targets for modulating dis- crete RGS-effector interactions (for review, see Hollinger and Hepler, 2002).…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2010

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Regulator of G protein signaling (RGS) proteins act to temporally modulate the activity of G protein subunits after G proteincoupled receptor activation. RGS proteins exert their effect by directly binding to the activated G␣ 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 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/G␣ interaction surface, modestly inhibits G␣ binding and GTPase acceleration. In addition, we report that modification of Cys148, a residue located on the opposite face of RGS4, can disrupt RGS/G␣ interaction through an allosteric mechanism that almost completely inhibits the G␣-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 G␣; 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 smallmolecule ligands.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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

et al. 2010

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“…Consequently, we tested CCG-63808 and CCG-63802 with a panel of RGS4 RGS domain cysteine mutants by FCPIA (Table 3). The G protein binding affinity of these RGS mutants has been described previously (Roof et al, 2009), and the K d values ranged from 3 to 12 nM, not drastically different from that of wild-type RGS4. No single cysteine could fully account for the effects of these compounds, but it seems that three cysteines (Cys148, Cys132, and Cys95) are important for full sensitivity to CCG-63808 and CCG-63802.…”

Section: Rgsmentioning

confidence: 99%

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

Blazer¹,

Roman²,

Chung³

et al. 2010

Mol Pharmacol

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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 [((, inhibit the interaction between RGS4 and G␣ o with an IC 50 value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 Ͼ 19 ϭ 16 Ͼ 8 Ͼ Ͼ 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to G␣ o . 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.

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“…Among possible molecules with potential therapeutic application are peptides that can be retrieved from a peptide library as reported by Roof et al (2009). The library screening allowed identifying an inhibitor called RGS4 (a regulator of G protein signaling).…”

Section: Low-abundance Proteome Discoverymentioning

confidence: 99%

Other Applications of Combinatorial Peptide Libraries

Righetti¹,

Boschetti

2013

Low-Abundance Proteome Discovery

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

Cited by 21 publications

References 11 publications

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

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

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

Other Applications of Combinatorial Peptide Libraries

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