Fig. 1

Identification of small-molecule OTUD3 inhibitor. A Diagram of human OTUD3, showing ovarian tumor (OTU) and ubiquitin-associated (UBA) domains, and active site (Cys76) directly involved in catalysis. B Crystal structure of the OTUD3 OTU domain. A cartoon representation is shown. The S1 Ub-binding site, Cys76, 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 3TMP. 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 A549 and H1299 treated with different candidate drugs at the indicated concentration (50 μM) for 72 h and cell survival rate was detected by CCK-8 assays. G A549 cells treated with DMSO, Estradiol Cypionate and Rolapitant at the indicated concentrations (15–25 μM) for 0–96 h. Cisplatin (10 μM) was used as a positive control. F, G The results were presented as the mean ± SD from 3 independent experiments. (n = 3; *, p < 0.05; **, p < 0.01, ***, p < 0.001 vs. DMSO). H MST (microscale thermophoresis) assays were uesd to detect the interaction of OTUD3 with Rolapitant and Estradiol Cypionate in vitro. I Chemical structure of Rolapitant. J Surface representation of the structure of the OTUD3 OTU domain in complex with Rolapitant. The residues around Rolapitant are highlighted in violet. K Overall structure of OTUD3 OTU domain in complex with Rolapitant, and Close-up view of the compound binding site highlighting key residues. OTUD3: green cartoon; Rolapitant: orange sticks; OTUD3 (Asn136, Ile152, Trp160, Ile162, Tyr177 and Tyr183) side chains are represented as sticks. Dashed lines showing the key interactions between OTUD3 and Rolapitant. Blue dashed lines represent hydrogen bonds. Green dashed lines indicate π-stacking interactions. Grey dashed lines indicate hydrophobic interactions. Binding site detail, showing the interactions between Rolapitant and OTU domain residues. Hydrogen bonds are formed between Asn136 and Rolapitant. π-stacking interactions are formed between Tyr177 and Rolapitant. Hydrophobic interactions are formed between Ile152, Trp160, Ile162, Tyr177, Tyr183 and Rolapitant. The PLIP package was used to analyze protein-ligand interactions. B, C, D, J,and K The images were generated with The PyMOL Molecular Graphics System (version 2.6.0a0)