title: Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay.
abstract: 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.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Storaska
authors.forename: Jian P
authors.initials: JP
authors.lastname: Mei
authors.forename: Meng
authors.initials: M
authors.lastname: Wu
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Susan M
authors.initials: SM
authors.lastname: Wade
authors.forename: Levi L
authors.initials: LL
authors.lastname: Blazer
authors.forename: Benita
authors.initials: B
authors.lastname: Sjögren
authors.forename: Corey R
authors.initials: CR
authors.lastname: Hopkins
authors.forename: Craig W
authors.initials: CW
authors.lastname: Lindsley
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Lin
authors.forename: Joseph J
authors.initials: JJ
authors.lastname: Babcock
authors.forename: Owen B
authors.initials: OB
authors.lastname: McManus
authors.forename: Richard R
authors.initials: RR
authors.lastname: Neubig
mesh_terms: D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D020710:RGS Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D020710:RGS Proteins; D043587:Receptor, Muscarinic M3; D054852:Small Molecule Libraries; C098799:RGS4 protein; D019204:GTP-Binding Proteins; D002118:Calcium
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
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