Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay

Andrew J. Storaska, Jian P. Mei, Meng Wu, Min Li, Susan M. Wade, Levi L. Blazer, Benita Sjögren, Corey R. Hopkins, Craig W. Lindsley, Zhihong Lin, Joseph J. Babcock, Owen B. McManus, Richard R. Neubig

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


Regulator of G-protein signaling (RGS) proteins potently suppress G-protein coupled receptor (GPCR) signal transduction by accelerating GTP hydrolysis on activated heterotrimeric G-protein α subunits. RGS4 is enriched in the CNS and is proposed as a therapeutic target for treatment of neuropathological states including epilepsy and Parkinson's disease. Therefore, identification of novel RGS4 inhibitors is of interest. An HEK293-FlpIn cell-line stably expressing M3-muscarinic receptor with doxycycline-regulated RGS4 expression was employed to identify compounds that inhibit RGS4-mediated suppression of M3-muscarinic receptor signaling. Over 300,000 compounds were screened for an ability to enhance Gαq-mediated calcium signaling in the presence of RGS4. Compounds that modulated the calcium response in a counter-screen in the absence of RGS4 were not pursued. Of the 1365 RGS4-dependent primary screen hits, thirteen compounds directly target the RGS-G-protein interaction in purified systems. All thirteen compounds lose activity against an RGS4 mutant lacking cysteines, indicating that covalent modification of free thiol groups on RGS4 is a common mechanism. Four compounds produce >85% inhibition of RGS4-G-protein binding at 100μM, yet are >50% reversible within a ten-minute time frame. The four reversible compounds significantly alter the thermal melting temperature of RGS4, but not G-protein, indicating that inhibition is occurring through interaction with the RGS protein. The HEK cell-line employed for this study provides a powerful tool for efficiently identifying RGS-specific modulators within the context of a GPCR signaling pathway. As a result, several new reversible, cell-active RGS4 inhibitors have been identified for use in future biological studies.

Original languageEnglish (US)
Pages (from-to)2848-2855
Number of pages8
JournalCellular Signalling
Issue number12
StatePublished - Dec 2013
Externally publishedYes


  • G-protein coupled receptors
  • High-throughput screen
  • M muscarinic acetylcholine receptor
  • Regulator of G-protein signaling
  • Small molecule inhibitor

ASJC Scopus subject areas

  • Cell Biology


Dive into the research topics of 'Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay'. Together they form a unique fingerprint.

Cite this