Reversible, allosteric small-molecule inhibitors of regulator of G protein signaling proteins

Abstract

Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((2E)-2-(1,3-benzothiazol-2-yl)-3-[9-methyl-2-(3-methylphenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]prop-2-enenitrile)], inhibit the interaction between RGS4 and Galpha(o) with an IC(50) value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 > 19 = 16 > 8 >> 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to Galpha(o). On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.

Fig. 1.

Characterization of the RGS4 TR-FRET high-throughput assay. A, schematic of RGS4-Gα_o TR-FRET assay. Gα_o is labeled with the LanthaScreen Tb-chelate donor fluorophore, and RGS4 is labeled with an Alexa Fluor 488 acceptor fluorophore. Excitation and emission maxima are listed for each fluorophore. B, representative data showing the AlF₄⁻/GDP dependence of the interaction between RGS4-AF488 and 10 nM Tb-Gα_o. This saturable interaction has a K_d of 35 ± 4 nM. C, two compounds identified in the high-throughput screen, CCG-63802 and CCG-63808, dose-dependently inhibit the TR-FRET signal between RGS4-AF488 and Tb-Gα_o with IC₅₀ values of 1.4 (0.76; 2.6 μM) and 1.9 μM (1.02; 3.5 μM), respectively. Data (n = 3 for all data) are presented as mean ± S.E.M. or mean (95% confidence interval) in B and C, respectively. D, the chemical structures of CCG-63802 and CCG-63808.

Fig. 2.

RGS specificity of CCG-63802 (A) and CCG-63808 (B) determined by multiplex FCPIA analysis (n ≥ 3). RGS-coated beads were treated with the indicated concentration of compound for 15 min at room temperature, after which GDP/AlF₄-bound Gα_o-AF532 was added and allowed to incubate with the RGS/compound mixture for 30 min before analysis. All data were calculated by using nonlinear least-squares regression with the bottom of the curves constrained to 0% binding. Data are presented as mean ± S.E.M. from at least three separate experiments.

Fig. 3.

Single-turnover GAP analysis of small-molecule RGS inhibitors with RGS4. A, RGS4 treated with 100 μM CCG-4986, CCG-63808, or CCG-63802 lacks the ability to increase the intrinsic hydrolysis rate of Gα_o. Representative GAP data are shown. All experiments were performed a minimum of three times. B, rate constants of GTP hydrolysis. Rate constants are presented as mean ± S.E.M. from at least three independent experiments. ***, p < 0.001 versus the DMSO-treated RGS control.

Fig. 4.

CCG-63802 specifically binds to RGS4 and not to Gα_o. A, purified RGS4 shows a dose-dependent change in melting temperature in the presence of CCG-63802 (EC₅₀ ∼26 μM). B, a saturating concentration of CCG-63802 (100 μM) does not affect the melting temperature of Gα_o. Data are presented as mean ± S.E.M. of three separate experiments.

Fig. 5.

CCG-63802 and CCG-63808 are reversible inhibitors of RGS4 (A) and RGS19 (B). CCG-4986 is an irreversible inhibitor of RGS4 and RGS19. In all cases, RGS-coated FCPIA beads were treated with 50 μM compound (or vehicle, DMSO) and then extensively washed. The beads were then split into two groups and tested for the ability to interact with Gα_o-AF532 in the presence or absence of 50 μM compound. Data shown are the mean ± S.E.M. of three separate experiments.

Fig. 6.

CCG-63802 is less sensitive to glutathione than other RGS4 inhibitors. A and B, CCG-63802 (A) and CCG-63808 (B) retain full inhibitory activity in the presence of 2 mM glutathione. The potency is right-shifted by approximately 0.5–1 Log (CCG-63802: Log IC₅₀ −5.25 ± 0.07 to −4.39 ± 0.07; CCG-63808: Log IC₅₀ −5.39 ± 0.06 to −4.68 ± 0.03). n = 2. C, in contrast, CCG-4986 loses more than two logs of potency (Log IC₅₀ −5.87 ± 0.03 to −3.66 ± 0.15) in the presence of glutathione. n = 3. Data presented as mean ± S.E.M.

Fig. 7.

CCG-63802 and CCG-63808 inhibit the GAP activity of a cysteine-null RGS4 mutant. A, CCG-63802 or CCG-63808 (100 μM) inhibits the ability of RGS4c to accelerate the rate of GTP hydrolysis by Gα_o. Representative data are shown. All experiments were performed a minimum of three times. B, rate constants of GTP hydrolysis. Rate constants are presented as mean ± S.E.M. from at least three independent experiments. **, p < 0.01 versus the DMSO-treated RGS control.