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.

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.

Figure 1

Inhibitors targeting RGS4 enhance carbachol-stimulated calcium signaling in cells treated with Doxycycline. (A) HEK-FlpIn cells stably transfected with M₃-muscarinic receptor produce a calcium transient in response to carbachol. Overnight treatment with Doxycycline (Dox) markedly suppresses the Gα_q-mediated calcium response through induction of RGS4 protein. (B) HA-tagged RGS4 is minimally detectable in HEK-FlpIn cells prior to treatment with Dox. Probing with an anti-HA antibody after a 45 hour treatment with Dox shows a marked increase in RGS4 protein, producing levels similar to transiently transfected cells (HEK Trans). (C) Representative primary screening hits that increase the carbachol-stimulated calcium transient towards the −Dox control (open squares) by antagonizing RGS4-mediated suppression of Gα_q signaling. The primary screening hits were tested in duplicate wells at 10 μM (final). Carb, carbachol; Dox, Doxycycline; HEK Trans, transiently transfected cells; HEK Mock, mock transfection with empty pCDNA vector.

Figure 2

Activity summary of the compounds tested on RGS4 in FCPIA and SS-GAP. All 58 primary screen hits were assayed for an ability to block RGS4-Gα_o equilibrium binding in FCPIA and inhibit RGS4-stimulation of Gα_i1 nucleotide hydrolysis in SS-GAP. Compounds with an IC₅₀ value less than 100 μM were selected for further analysis, resulting in 27 compounds meeting this criterion in SS-GAP and 13 in FCPIA. All 13 compounds active in (A) FCPIA were also active in (B) SS-GAP. The IC₅₀ values range from approximately 2-55 μM in both assays. CID 1472216 is the most potent RGS4 inhibitor tested, IC₅₀= 1.6±0.4 μM in SS-GAP and 1.7±0.5 μM in FCPIA. The data are the mean ± S.E.M. of three independent experiments in duplicate wells. SS-GAP, steady-state GTPase acceleration protein assay; FCPIA, flow cytometry protein interaction assay; pIC₅₀, log IC_50.

Figure 3

Determination of RGS4 inhibitor reversibility using FCPIA. RGS4-labeled Luminex beads were treated with 100 μM compound or DMSO for 30 minutes at room temperature before washing the beads three times. The beads were then split in two sets with DMSO or 100 μM compound added back to the no wash set (open bar). Both treated and washed RGS4-coupled beads were tested for inhibition of Gα_o binding using FCPIA. Statistics: One-Way ANOVA with Bonferroni’s multiple comparisons *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are the result of three independent experiments ± S.E.M.

Figure 4

The reversible RGS4 inhibitors alter the thermal melting temperature of RGS4, but not Gα. RGS4 and Gα_i1 were treated with 100μM compound or DMSO for 30 minutes prior to measuring thermal stability changes of each protein across a temperature gradient of 30-90°C. Melting was monitored as an increase in 1,8 ANS fluorescence using a Thermofluor instrument. Data are plotted as temperature change relative to the DMSO control for each protein. None of the compounds significantly changed the thermal stability of Gα_i1 compared to DMSO control. Statistics: One-Way ANOVA with Bonferroni’s multiple comparisons *, P < 0.05; **, P < 0.01; ***, P < 0.001. Data are the result of three independent experiments ± S.E.M.

Figure 5

Compounds 1905297 and 1777233 show selectivity for RGS4 over four other RGS isoforms tested in FCPIA. (A) Compound 1905297 is at least 3.6-fold selective for RGS4 over all other RGS proteins tested. (B) Compound 1777233 is slightly more potent towards RGS4, although there is only a 2-fold potency difference from RGS19 and 5-fold from RGS16. (C) & (D) Both 5428579 and 1389577 have approximately equal potency towards RGS4 and RGS19. In addition, activity was observed against RGS7 and RGS16 for 5428579 and RGS7 for 1389577. Compounds were tested from 100 nM to 100 μM and data are the mean of three independent experiments.

Figure 6

Cellular activity of the four reversible RGS4 inhibitors. (A) 1905297 increased the calcium response over Dox-treated control cells at 30 and 10 μM, although effects at lower concentrations may be mitigated as a result of lower overall carbachol response. (B)–(D) The carbachol response is increased over Dox-treated control cells at 1 μM compound and above. Data shown are representative responses performed in duplicate wells.