AtomNet-Aided OTUD7B Inhibitor Discovery and Validation

Abstract

Protein deubiquitinases play critical pathophysiological roles in cancer. Among all deubiquitinases, an oncogenic function for OTUD7B has been established in genetic NSCLC murine models. However, few deubiquitinase inhibitors have been developed due to technical challenges. Here, we report a putative small molecule OTUD7B inhibitor obtained from an AI-aided screen of a 4 million compound library. We validated the effects of the OTUD7B inhibitor (7Bi) in reducing Akt-pS473 signals in multiple NSCLC and HEK293 cells by blocking OTUD7B-governed GβL deubiquitination in cells, as well as inhibiting OTUD7B-mediated cleavage of K11-linked di-ub in an in vitro enzyme assay. Furthermore, we report in leukemia cells, either genetic depletion or 7Bi-mediated pharmacological inhibition of OTUD7B reduces Akt-pS473 via inhibiting the OTUD7B/GβL signaling axis. Together, our study identifies the first putative OTUD7B inhibitor showing activities both in cells and in vitro, with promising applications as a therapeutic agent in treating cancer with OTUD7B overexpression.

Keywords: NSCLC; OTUD7B; cell proliferation; leukemia; small molecule inhibitor.

Figure 1

Searching for OTUD7B small molecule inhibitors. (A) A cartoon illustration for the pipeline of searching for OTUD7B small molecule inhibitors. AtomNet was used to search a 4 million small molecule library for hits fitting into the OTUD7B-OTU structure. Top hits were purchased and effects of OTUD7B inhibition by these small molecules were tested by both Akt-pS473 ELISA and Western blotting in A549 cells. Overlapping hits from two assays were identified and tested further. (B) Immunoblot (IB) analyses of whole cell lysates (WCL) derived from control or OTUD7B depleted A549 cells. (C) Representative colony formation analyses images using A549 cells obtained in (B) and quantified in (D). Error bars were calculated as mean+/-SD, n = 3. * p < 0.05 (one-way ANOVA test). (E) A heatmap illustration of Akt-pS473 ELISA results obtained from treating A549 cells with 10 μM of each indicated chemical for 16 h. (F) A list of chemicals with the ability to suppress 30% Akt-pS473 signals from assays in (E). (G) IB analyses of WCL from A549 cells treated with indicated compounds for 16 h. (H) A list of chemicals with the ability to suppress 10% Akt-pS473 signals from assays in (G). (I) A list of overlapped hits from both (F,H).

Figure 2

Validation for OTUD7B small molecule inhibitors in cells. (A–D) IB analyses of WCL from indicated NSCLC cells treated with 10 μM of indicated compounds for 16 h. (E,F) IB analyses of WCL from either A549 (E) or HEK293 (F) cells treated with indicated doses of compound #19 for 16 h. (G) IB analyses of Ni-NTA pulldowns and WCL from HEK293 cells transfected with indicated DNA constructs. Where indicated, cells were treated with 10 μM of indicated compounds for 16 h before cell collection. (H–L) Quantifications of cell viability by MTT assays in A549 cells treated with indicated doses of indicated compounds for 72 h. Error bars were calculated as mean ± SD, n = 3. * p < 0.05 (one-way ANOVA test).

Figure 3

7Bi inhibits OTUD7B activity in vitro and suppress cell growth in NSCLC cells. (A,B) A representative gel image (A) for the in vitro OTUD7B deubiquitination assay incubating 5 nM of recombinant OTUD7B with 0.5 μM of K11-linked di-ub for 10 min in the presence of indicated doses of 7Bi. Mono-ub was quantified for IC50 value determination (B). (C,D) A representative gel image (C) for the in vitro USP21 deubiquitination assays incubating 80 nM of recombinant active USP21 proteins with 0.5 μM of K11-linked di-ub for 10 min in the presence of indicated doses of 7Bi. Mono-ub was quantified for IC50 value determination (D). (E–H) In vitro deubiquitination assays using ubiquitin-AMC as a substrate and indicated deubiquitinases. Normalized reading vs. reaction time periods was plotted. (I) A representative gel image for the in vitro OTUD7B deubiquitination assay incubating 5 nM of recombinant active OTUD7B proteins with 0.5 μM of K11-linked di-ub for 10 min in the presence of 100 μM of 7Bi (from either Mcule (90% purity) or recrystallized (99% purity)). (J) IB analysis of WCL derived from HEK293 cells treated with 10 μM of 7Bi (from either Mcule (90% purity) or recrystallization-purified (99% purity)). (K) IB analyses of GβL-IP and WCL from A549 cells treated with control or 10 μM 7Bi for 16 h. (L) IB analyses of HA-IP and WCL from HEK293 cells transfected with indicated DNA constructs. Where indicated, indicated doses of 7Bi were used to treat cells for 16 h before cell collection. (M) Representative colony formation images from indicated NSCLC cells treated with indicated doses of 7Bi once for 10 days and quantified in (N–P). Error bars were calculated as mean ± SD, n = 3. * p < 0.05 (one-way ANOVA test). (Q) Quantifications of cell viability by MTT assays in HEK293 cells treated with indicated doses of indicated compounds for 72 h. Error bars were calculated as mean ± SD, n = 3. * p < 0.05 (one-way ANOVA test).

Figure 4

OTUD7B inactivation suppresses leukemia cell proliferation. (A–C) IB analyses of WCL from indicated cells with depletion of OTUD7B by shRNAs. (D) IB analyses of GβL-IP and WCL from indicated K562 cells. (E–G) Quantifications of cell viability by MTT assays in indicated cells treated with indicated doses of indicated compounds for 72 h. Error bars were calculated as mean ± SD, n = 3. * p < 0.05 (one-way ANOVA test). (H–J) IB analyses of WCL from indicated cells treated with indicated doses of 7Bi for 16 h. (K) IB analyses of GβL-IP and WCL from K562 cells treated with control or 10 μM 7Bi for 16 h. (L–N) Quantifications of cell viability by MTT assays in indicated cells treated with indicated doses of indicated compounds for 72 h. Error bars were calculated as mean ± SD, n = 3. * p < 0.05 (one-way ANOVA test).