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A Nanomolar-Potency Small Molecule Inhibitor of Regulator of G-Protein Signaling Proteins

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† ‡ § Department of Pharmacology, Department of Internal Medicine (Cardiovascular Medicine), and §Center for Chemical Genomics, The University of Michigan, Ann Arbor, Michigan 48109, United States
Department of Pharmacy and Pharmacology, University of Bath, Bath, United Kingdom
*Department of Pharmacology, University of Michigan, 1301 MSRBIII, 1150 W. Medical Center Dr., Ann Arbor, MI 41809. Phone: (734) 764-8165. Fax: (734) 763-4450. E-mail: [email protected]
Cite this: Biochemistry 2011, 50, 15, 3181–3192
Publication Date (Web):February 17, 2011
Copyright © 2011 American Chemical Society

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    Regulators of G-protein signaling (RGS) proteins are potent negative modulators of signal transduction through G-protein-coupled receptors. They function by binding to activated (GTP-bound) Gα subunits and accelerating the rate of GTP hydrolysis. Modulation of RGS activity by small molecules is an attractive mechanism for fine-tuning GPCR signaling for therapeutic and research purposes. Here we describe the pharmacologic properties and mechanism of action of CCG-50014, the most potent small molecule RGS inhibitor to date. It has an IC50 for RGS4 of 30 nM and is >20-fold selective for RGS4 over other RGS proteins. CCG-50014 binds covalently to the RGS, forming an adduct on two cysteine residues located in an allosteric regulatory site. It is not a general cysteine alkylator as it does not inhibit activity of the cysteine protease papain at concentrations >3000-fold higher than those required to inhibit RGS4 function. It is also >1000-fold more potent as an RGS4 inhibitor than are the cysteine alkylators N-ethylmaleimide and iodoacetamide. Analysis of the cysteine reactivity of the compound shows that compound binding to Cys107 in RGS8 inhibits Gα binding in a manner that can be reversed by cleavage of the compound−RGS disulfide bond. If the compound reacts with Cys160 in RGS8, the adduct induces RGS denaturation, and activity cannot be restored by removal of the compound. The high potency and good selectivity of CCG-50014 make it a useful tool for studying the functional roles of RGS4.

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    A table containing the chemical structure and RGS inhibitory activity of CCG-50014 and CCG-303778, an analogue of CCG-50014 in which the sulfur atom is replaced with a methylene, and a figure depicting the rate constants of GTP hydrolysis by Gαo in the presence and absence of 100 μM CCG-50014. This material is available free of charge via the Internet at

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