Screen Targeting Lung and Prostate Cancer Oncogene Identifies Novel Inhibitors of RGS17 and Problematic Chemical Substructures

Abstract

Regulator of G protein signaling (RGS) proteins temporally regulate heterotrimeric G protein signaling cascades elicited by G protein-coupled receptor activation and thus are essential for cell homeostasis. The dysregulation of RGS protein expression has been linked to several pathologies, spurring discovery efforts to identify small-molecule inhibitors of these proteins. Presented here are the results of a high-throughput screening (HTS) campaign targeting RGS17, an RGS protein reported to be inappropriately upregulated in several cancers. A screen of over 60,000 small molecules led to the identification of five hit compounds that inhibit the RGS17-Gα_o protein-protein interaction. Chemical and biochemical characterization demonstrated that three of these hits inhibited the interaction through the decomposition of parent compound into reactive products under normal chemical library storage/usage conditions. Compound substructures susceptible to decomposition are reported and the decomposition process characterized, adding to the armamentarium of tools available to the screening field, allowing for the conservation of resources in follow-up efforts and more efficient identification of potentially decomposed compounds. Finally, analogues of one hit compound were tested, and the results establish the first ever structure-activity relationship (SAR) profile for a small-molecule inhibitor of RGS17.

Keywords: AlphaScreen; RGS proteins; SAR; compound decomposition; high-throughput screening.

Figure 1 –

Structures of the five lead compounds selected for characterization based on initial follow up. Compounds will be referred to in text by their respective numeric identifiers, I–V.

Figure 2 –

A) Crystal structure of RGS17 (PDB - 1ZV4). G protein interaction interface is noted, and the allosteric Cys 117 is represented as spheres in yellow. B–F) Dose response analysis of the 5 lead compounds against WT and mutant protein. WT IC₅₀ values were 9.5 µM, 19.2µM, 14.4 µM, 31.1 µM, and 14.3 µM for panels B–F respectively. No compound inhibited mutant RGS17 (C117A) by the highest concentration tested (100 µM). WT data are n=3 in duplicate, mutant data are n=2 in duplicate. All data shown as mean ± SEM.

Figure 3 -

Three lead compounds decompose in DMSO. Compounds are depicted above their representative dose response analysis. Initial from library data are n=3 in duplicate. All other data are n=2 in duplicate. All dose response data shown as mean ± SEM. Panels G-I are the thiol reactivity results for the compound products over time. All data normalized to NAC alone (negative control), NEM represents positive control. Significance calculated via one way ANOVA. Holm-Sidak multiple comparison analysis shown on graph with respect to NAC control (*) and with respect to t=0 (#). P-values are */# - P ≤ 0.05, **/## - P ≤ 0.01, ***/### - P < 0.001. Thiol reactivity data are n=3.

Figure 4 -

Analogs of Compound II tested in Figure 5.

Figure 5 -

(A and B) Dose response analysis of analogs depicted in Figure 4. IC₅₀ values listed in Table SV. Data are n=3 in duplicate shown as mean ± SD. (C) Thiol reactivity of Compound II, Compound II-3, and Compound III. All data normalized to NAC alone (negative control), NEM represents positive control. A statistically significant difference between the groups was determined via one way ANOVA (F(4,13) = 6.376, p = 0.05). Holm-Sidak multiple comparison analysis shown on graph with respect to NAC control (*). Thiol reactivity data are n=3.

Figure 6 –

Compound II, II-3, and III were tested for selective inhibition of RGS17 over RGS7, RGS10, and RGS18. For all three compounds, RGS17 was the most potently inhibited, with a statistically significant reduction in signal compared to the inhibition observed for RGS7, RGS10, and RGS18. Significance determined via one way ANOVA with Holm-Sidak multiple comparison post hoc analysis. Significance shown on graph are the multiple comparison analysis with respect to RGS17, *** - P < 0.001. Data are n=3 in duplicate ± SD.