Mechanism of Action and Structural Requirements of Constrained Peptide Inhibitors of RGS Proteins

Roof, Rebecca A.; Jin, Yijun; Roman, David L.; Sunahara, Roger K.; Ishii, Masaru; Mosberg, Henry I.; Neubig, Richard R.

doi:10.1111/j.1747-0285.2006.00373.x

Cited by 27 publications

(40 citation statements)

References 30 publications

(58 reference statements)

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“…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%

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%

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

et al. 2010

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“…These studies led to several peptides that effectively blocked RGS protein activity in vitro (e.g. YJ34 and 5nd; Jin et al, 2004;Young et al, 2004;Roof et al, 2006;Wang et al, 2008). The first published small molecule RGS inhibitor, CCG-4986, was identified by the group of Richard Neubig, using a novel flow cytometry-based PPI assay (Roman et al, 2007).…”

Section: Advances In Rgs Protein Drug Discovery -From Biochemical Actmentioning

confidence: 99%

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

Sjögren

2017

British J Pharmacology

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Regulators of G protein signalling (RGS) proteins are celebrating the 20th anniversary of their discovery. The unveiling of this new family of negative regulators of G protein signalling in the mid-1990s solved a persistent conundrum in the G protein signalling field, in which the rate of deactivation of signalling cascades in vivo could not be replicated in exogenous systems. Since then, there has been tremendous advancement in the knowledge of RGS protein structure, function, regulation and their role as novel drug targets. RGS proteins play an important modulatory role through their GTPase-activating protein (GAP) activity at active, GTP-bound Gα subunits of heterotrimeric G proteins. They also possess many non-canonical functions not related to G protein signalling. Here, an update on the status of RGS proteins as drug targets is provided, highlighting advances that have led to the inclusion of RGS proteins in the IUPHAR/BPS Guide to PHARMACOLOGY database of drug targets.Abbreviations DEP, Disheveled Egl-10 Pleckstrin; GAP, GTPase-activating protein; GGL, G protein γ-like; PPI, protein-protein interaction; RGS, regulator of G protein signalling; R7BP, R7 binding protein; R9AP, RGS9 associated protein IntroductionG protein-mediated signalling pathways have played a pivotal role in drug discovery and development for many decades. The large family of GPCRs or their downstream effectors are the target of 40% of clinically used drugs and thus represent a multi-billion-dollar industry (Wise et al., 2002). Interestingly, of the more than 300 non-olfactory GPCRs known, only a fraction of them are targeted by drugs. Thus, there is a large untapped area of drug development still available. Moreover, many GPCR drugs are associated with low efficacy and/or side effects. More targeted therapies are therefore required, and as we learn more about the structure and function of GPCRs and their regulators, these goals will be achievable.All biological signals are tightly regulated and for every on-switch there is usually an off-switch. GPCRs are activated by ligands, transmitting signalling information to Gα subunits of heterotrimeric G proteins by enhancing the exchange of GDP for GTP in the Gα nucleotide binding site, which results in the dissociation of Gα from Gβγ dimers and activation of both G protein components. Deactivation of G proteins does not occur by simple reversal of nucleotide exchange, but rather by an independently regulated GTPase activity, hydrolyzing GTP to GDP. Although Gα proteins possess an intrinsic ability to hydrolyze GTP, this process is very slow and cannot account for the transient nature of intracellular signalling cascades in vivo. Hence, additional kinetic mechanisms are required for the physiological timing of signals. One of the most critical of these kinetic mechanisms is mediated through regulator of G protein signalling (RGS) proteins, which have received increasing interest as novel drug targets in the past two decades. As a result, RGS proteins have now been added as the most recent ad...

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“…Previous work from our laboratory identified a cyclic octapeptide derived from the RGS interaction site of G␣ i subunits that inhibits RGS4 and alters G protein-coupled inwardly rectifying potassium channel current kinetics in atrial cells (Jin et al, 2004;Roof et al, 2006). Another group performed a smallmolecule RGS inhibitor screen using a yeast two-hybrid readout (Young et al, 2004) that yielded some low-affinity inhibitors, but no structures or follow-up information was reported.…”

mentioning

confidence: 99%

“…Single-turnover GTP hydrolysis measurements in 96-well plates were performed as described previously (Roof et al, 2006). Compounds were preincubated with the RGS protein for 10 to 15 min on ice before initiating the GAP assay.…”

mentioning

confidence: 99%

Identification of Small-Molecule Inhibitors of RGS4 Using a High-Throughput Flow Cytometry Protein Interaction Assay

Roman¹,

Talbot²,

Roof³

et al. 2006

Mol Pharmacol

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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 ␣-subunits (G␣) and thus shorten the time course and reduce the magnitude of G-protein ␣-and ␤␥-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 G␣-RGS interactions in a high-throughput screen, we identified the first small-molecule inhibitor of an RGS protein. Of 3028 compounds screened, 1,sulfonyl]-4-nitrobenzenesulfinimidoate (CCG-4986), inhibited RGS4/G␣ o binding with 3 to 5 M potency. It binds to RGS4, inhibits RGS4 stimulation of G␣ o GTPase activity in vitro, and prevents RGS4 regulation of -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/G␣ protein-protein interactions with small molecules as a novel means to modulate GPCR-mediated signaling processes.

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Mechanism of Action and Structural Requirements of Constrained Peptide Inhibitors of RGS Proteins

Cited by 27 publications

References 30 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

The evolution of regulators of G protein signalling proteins as drug targets – 20 years in the making: IUPHAR Review 21

Identification of Small-Molecule Inhibitors of RGS4 Using a High-Throughput Flow Cytometry Protein Interaction Assay

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