Fig. 1: Identification of small-molecule OTUD3 inhibitors.
From: Discovery of an OTUD3 inhibitor for the treatment of non-small cell lung cancer
A Diagram of human OTUD3, showing ovarian tumor (OTU) and ubiquitin-associated (UBA) domains. B Crystal structure of the OTUD3 OTU domain. A cartoon representation is shown. The S1 Ub-binding site, N termini, and C termini are labeled. C Left: Crystal structure of the OTUD5 OTU domain (green) bound to Ub (orange) (PDB-ID:3TMP). Middle: Structure of the OTUD3 OTU domain (blue) superimposed to the OTUD5 OTU domain (green) bound to Ub (orange). Right: Approximate structure of the OTUD3 OTU domain (blue) bound to Ub (orange), Ub position was determined by sequence and structural alignment with OTUD5. D Identification of the docking GridBox that encloses the S1 Ub-binding site. E Schematic diagram of computational virtual screening based on molecular docking. F Cell proliferation assays showing that only compound 16 had a significant inhibitory effect on the proliferation of H1299 and H460 cell lines after 48 h of treatment. Data shown are mean ± SD. n = 5 independent experiments. Two-tailed unpaired Student’s t-test. ***p < 0.001, vs DMSO group. G Chemical structure of OTUDin3 (compound 16). H Surface representation of the structure of the OTUD3 OTU domain in complex with OTUDin3. The residues around OTUDin3 are highlighted in violet. I Overall structure of OTUD3 OTU domain in complex with OTUDin3, and Close-up view of the compound binding site highlighting key residues. OTUD3: skyblue cartoon; OTUDin3: yellow sticks; OTUD3 (Asn136, Gln154, Trp160, Ile162, Tyr177 and Tyr183) side chains are represented as sticks. Dashed lines showing the key interactions between OTUD3 and OTUDin3. Blue dashed lines represent hydrogen bonds. Green dashed lines indicate π-stacking interactions. Gray dashed lines indicate hydrophobic interactions. Binding site detail, showing the interactions between OTUDin3 and OTU domain residues. Hydrogen bonds are formed between Gln154, Trp160, Tyr177, Tyr183 and OTUDin3. π-stacking interactions are formed between Trp160, Tyr177 and OTUDin3. Hydrophobic interactions are formed between Asn136, Ile162 and OTUDin3. The PLIP package was used to analyze protein-ligand interactions. J Representative SPR sensorgram of OTUD3 OTU (aa 52-209) to measure affinity parameters of OTUDin3. The KD value was 0.32 μM. K OTUD3 OTU (aa 52-209) and K48-linked Di-Ubiquitin were incubated with indicated concentrations of OTUDin3 for 3 h at 37 °C, followed by western blotting with indicated antibodies. L Purified GST-OTUD3C76A and Di-Ubiquitin was incubated with OTUDin3 or DMSO. Proteins retained on Sepharose were blotted with the indicated antibodies, or visualized by SDS-PAGE and Coomassie blue staining. B, C, D, H, and I The images were generated with The PyMOL Molecular Graphics System (version 2.6.0a0). J–L All panels are representative results of three or more independent experiments.
