Abstract
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.
Footnotes
↵
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.This work was supported by the Michigan Chemistry-Biology Interface Training Program, which is funded through the National Institutes of Health National Institute of General Medical Sciences [Grant T32-GM00008597]; the National Institutes of Health National Institute on Drug Abuse [Grant R01-DA023252]; and the National Institutes of Health National Institute of General Medical Sciences [Grant F32-GM076821].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.065128.
-
ABBREVIATIONS:
- RGS
- regulator of G protein signaling
- TR-FRET
- time-resolved fluorescence resonance energy transfer
- FCPIA
- flow cytometry protein interaction assay
- SMPPII
- small-molecule protein-protein interaction inhibitor
- PPI
- protein-protein interaction
- GAP
- GTPase-accelerating protein
- GPCR
- G protein-coupled receptor
- DTT
- dithiothreitol
- MBP
- maltose-binding protein
- DMSO
- dimethyl sulfoxide
- PCR
- polymerase chain reaction
- BSA
- bovine serum albumin
- Tm
- melting temperature
- DRC
- dose-response curve
- 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)
- CCG-63808
- ((2E)-2-(1,3-benzothiazol-2-yl)-3-[9-methyl-2-(4-fluorolphenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]prop-2-enenitrile)
- CCG-4986
- methyl-N-[(4-chlorophenyl)sulfonyl]-4-nitrobenzenesulfinimidoate
- CI-1033
- N-[-4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-2-propenamide.
- Received March 29, 2010.
- Accepted June 15, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|
Log in using your username and password
Purchase access
In this issue
Reversible, Allosteric Small-Molecule Inhibitors of Regulator of G Protein Signaling Proteins
