Figure 1.
Scheme of Malachite Green Assay.
RGS protein interacts with Gαi and induces the hydrolysis of GTP to GDP, releasing free phosphate. In the presence of acid, molybdate releases water and complexes with the free phosphate. Lastly, the phosphomolybdate complex associates with the malachite green to produce a strong absorbance peak at 642 nm.
Figure 2.
Optimization of Malachite Green Assay for RGS4.
(A) Increasing concentrations of Gαi, 1 µM to 20 µM final, were compared using final concentrations of 200 nM RGS4 and 300 µM GTP. Absorbance at 642 nm was read every 10 min. Each sample was graphed as with 200 nM RGS4 (closed symbols) or without (open circles). GTP only (300 µM final) control wells were used for background subtraction. (B) Increasing concentrations of RGS4, from 50 to 400 nM final, were compared using Gαi at 5 µM final, and 300 µM GTP. Absorbance at 642 nm was read every 10 min. GTP only (150 µM final) control wells were used for background subtraction. (C) Increasing concentrations of the GTP, from 50 to 600 µM final, were compared using RGS4 (200 nM) and Gαi(5 µM), final concentrations. Samples were read at 642 nm absorbance every 10 min.
Figure 3.
Characterization of Malachite Green Assay with RGS8 and RGS17.
(A) Increasing concentrations of RGS8 from 5 nM final to 200 nM final, represented as closed symbols, show signal about equal to 2× the concentration of RGS4, similar to as shown in literature [4]. For comparison, 5 µM final Gαi was included, represented by open symbols. GTP only (150 µM final) control wells were used for background subtraction. (B) Using a Gαi double mutant protein with an accelerated Koff for GDP exchange and decrease Kcat for GTPase activity we can monitor the effect of RGS17 on the intrinsic GTPase activity of the Gαi subunit. GTP only (300 µM final) control wells were used for background subtraction.
Figure 4.
Determination of the Z-factor for 384-well and 1536-well assay.
(A) In a 384-well plate, 192 wells were used as a negative control (buffer only), represented by closed circles. An additional 192 wells were used as positive controls and were treated with CCG-50014, a potent RGS4 inhibitor, (10 µM final) represented by open circles [29]. The solid lines represent the mean value for the negative control and the positive control (1.74 and 0.92 respectively). The dashed lines marks the 3 standard deviation cut off for both the positive and negative control (standard deviation of 0.033 and 0.021 respectively). (B) This assay was conducted in 5.5% DMSO to mimic the actual concentration of DMSO in the pilot screen. In a 1536-well plate, 128 wells received buffer, negative control (closed symbols) and the remaining 128 wells received 10 µM final CCG-50014, positive control (open symbols). The solid lines represent the mean value for the negative control and the positive control (0.67 and 0.30 respectively). The dashed lines marks the 3 standard deviation cut off for both the positive and negative control (standard deviation of 0.028 and 0.021 respectively).
Figure 5.
Solid line represents mean negative control. Dashed line represents 3 standard deviations from control and consideration as a hit. (A) In plate one, 16 compounds were identified as initial hits. (B) In plate 2, an additional 43 compounds were identified as initial hits.
Figure 6.
Single Point Hit Confirmation and Control Screens.
(A) Single point hit confirmation assay was an analysis of each of the initial hits in a 384-well format (40 µM final for each compound). 7 compounds fell within 3 standard deviations of the negative control and were excluded from further analysis. (B) Phosphate control assay was a comparison of each compound’s (40 µM final) ability to inhibit the assay itself, containing 50 µM phosphate instead of protein. Dashed line represents 3 standard deviations from the negative control. 1 compound fell below 3 standard deviations and was excluded from further analysis. (C) At 40 µM final for each compound, the Gαi control assay evaluated each compound for inhibition of Gαi (5 µM final). The dashed line represents 3 standard deviations below the negative control. 5 compounds fell below 3 standard deviations and were excluded from further analysis. Filled bars represent compounds carried over to following experiments. Open bars represent compounds excluded from further analysis.
Figure 7.
ALPHA-Screen orthogonal assay and RGS4(Δ7) counter screen.
(A) At 40 µM final for each compound, this assay was used to confirm each compound as an inhibitor of RGS4 (20 nM final) through another assay. The dashed line represents the cutoff, 3 standard deviations from negative control. 15 compounds fell within 3 standard deviations of the negative control and were excluded from further analysis. (B) This single point assay, at 40 µM compound, was used to confirm activity of each compound against the RGS4(Δ7) mutant (200 nM final). The dashed line represents 25% inhibition, the cutoff for compounds carried to dose-response analysis. 18 compounds failed to inhibit the RGS4(Δ7) mutant of RGS4 and were excluded from further analysis. Filled bars represent compounds carried over to following experiments. Open bars represent compounds excluded from further analysis.
Figure 8.
Dose-response anaylsis of UI-5, UI-1590, UI-1907, UI-2034.
(A) Inceasing concentrations of compound challenged against RGS4(WT), 200 nM final, in the malachite green assay. (B) The same compounds were compared against the RGS4(Δ7) mutant. All compounds have marked lower potency against the RGS4(Δ7) than the RGS4(WT).
Figure 9.
Structure of identified Compounds.
(A) UI-5, also known as sanguinarium sulfate. (B) UI-1590 is the pre-therapeutic anti-cancer compound celastrol [40]. (C) UI-1907 is gambogic acid. (D) UI-2034, acetyl-isogambogic acid, is an analogue of UI-1907.
Figure 10.
Pilot screen results for Spectrum Library.
From the 2320 compound library, 59 compounds (2.5%) were considered hits. 52 of those compounds were confirmed in a single point assay. 6 compounds were found to inhibit either the assay or the Gαi directly, leaving 46 compounds (2.0%). An additional 15 compounds were removed for failing the ALPHA Screen orthogonal assay. And finally, 18 compounds were found to not inhibit RGS4(Δ7) at least 25% in the single point counter screen, leaving 13 compounds (0.6%).