Reversible inhibitors of regulators of G-protein signaling identified in a high-throughput cell-based calcium signaling assay |
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| Authors: | 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 |
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| Affiliation: | 1. Department of Pharmacology, University of Michigan, Ann Arbor, MI 48109, USA;2. Johns Hopkins Ion Channel Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA;3. Michigan State University, Department of Pharmacology & Toxicology, East Lansing, MI 48824, USA;4. Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA;5. Department of Pharmacology, Vanderbilt University, Nashville, TN 37232, USA;6. Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville, TN 37232, USA;7. Vanderbilt Specialized Chemistry Center for Accelerated Probe Development, Vanderbilt University Medical Center, Nashville, TN 37232, USA;8. Center for Chemical Genomics, University of Michigan, Ann Arbor, MI 48109, USA |
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| 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 100 μM, 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. |
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