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

Cell Signal. 2013 Dec;25(12):2848-55. doi: 10.1016/j.cellsig.2013.09.007. Epub 2013 Sep 14.


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.

Keywords: BSA; DMSO; FCPIA; G-protein coupled receptors; GAP; GTPase accelerating protein; High-throughput screen; M(3) muscarinic acetylcholine receptor; MBP; PPI; RGS; Regulator of G-protein signaling; Small molecule inhibitor; T(m); bovine serum albumin; dimethyl sulfoxide; flow cytometry protein interaction assay; maltose binding protein; melting temperature; protein–protein interaction; regulator of G-protein signaling.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Calcium / metabolism
  • Calcium Signaling / drug effects*
  • Cell Line
  • Drug Evaluation, Preclinical
  • GTP-Binding Proteins / antagonists & inhibitors*
  • GTP-Binding Proteins / metabolism
  • High-Throughput Screening Assays
  • Humans
  • RGS Proteins / antagonists & inhibitors
  • RGS Proteins / metabolism
  • Receptor, Muscarinic M3 / metabolism
  • Signal Transduction / drug effects
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology*


  • RGS Proteins
  • Receptor, Muscarinic M3
  • Small Molecule Libraries
  • RGS4 protein
  • GTP-Binding Proteins
  • Calcium