Cellular Validation of a Chemically Improved Inhibitor Identifies Monoubiquitination on OTUB2

Ubiquitin thioesterase OTUB2, a cysteine protease from the ovarian tumor (OTU) deubiquitinase superfamily, is often overexpressed during tumor progression and metastasis. Development of OTUB2 inhibitors is therefore believed to be therapeutically important, yet potent and selective small-molecule inhibitors targeting OTUB2 are scarce. Here, we describe the development of an improved OTUB2 inhibitor, LN5P45, comprising a chloroacethydrazide moiety that covalently reacts to the active-site cysteine residue. LN5P45 shows outstanding target engagement and proteome-wide selectivity in living cells. Importantly, LN5P45 as well as other OTUB2 inhibitors strongly induce monoubiquitination of OTUB2 on lysine 31. We present a route to future OTUB2-related therapeutics and have shown that the OTUB2 inhibitor developed in this study can help to uncover new aspects of the related biology and open new questions regarding the understanding of OTUB2 regulation at the post-translational modification level.


Contents Supplementary Schemes, Figures and Tables Page
Table S1.Overview of the purified recombinant human DUBs S13 Table S2.X-ray data processing and refinement statistics S14 Table S3.Primers sequences for site-directed mutagenesis S15  -S13-  -S15-Table S3.Primers sequences used for site-directed mutagenesis of OTUB2 K12R, K31R, K37R, K44R, K46R and K211R.C. The crude hydrazide was taken up in DCM (ca.0.01 -0.05M).Et3N (3 eq.) was added, followed by chloroacetylchloride (1.1 eq.)After TLC analysis indicated the reaction to be complete the mixture was diluted ca.2x with DCM and washed with an equal amount of H2O.The organic layer was dried (MgSO4), filtered through a paper filter and adsorbed onto Celite.Purification was performed using a silica gel column with an ethyl acetate/heptane gradient as eluent after which the purest fractions were combined and concentrated in vacuo at 40 °C.

D. Enzymatic Cyclopropanation Reactions. Preparation of the cells expressing
engineered myoglobins was done according to literature precedence. 4,5 reshly prepared cell pallets were washed in KPi buffer (50 mM, pH 7, 3x 25 mL) and concentrated to ~OD600=100-120 (cell stock solution).Then, a round-bottom flask was charged with an appropriate amount of KPi buffer which was subsequently degassed with N2 for 10 minutes.Under positive N2 pressure, an appropriate amount of cell stock solution was added to the flask to obtain a final cell concentration of OD600=20.The head space of the resulting solution was degassed under N2 for 10 minutes.A solution with the appropriate olefin in EtOH (1 eq., 10 mM final conc.) was added via syringe through a septum under positive N2 pressure.The reactions were initiated by the slow addition of an EDA solution in EtOH (3 eq., 30 mM final conc.) using a syringe pump over 3 hours.The reaction was stirred overnight at room temperature under positive N2 pressure.The crude product was extracted with DCM (3x 50 mL), dried over Na2SO4, filtrated, and concentrated under reduced pressure.The crude residue was purified by flash chromatography (hexanes/EtOAc, 95:5) and concentrated in vacuo.

Figure S1 .Figure S2 .Figure S4 .
Figure S1.Biochemical inhibition data linked to inhibition values in Table 1.(A) IC50 curves for compounds 1-10 after 2h incubation.(B) Determination of the kinact/KI values for compounds 5-10.Activity curves on the left and kobs vs inhibitor concentration on the right.

Figure S5 .
Figure S5.Gel-based competition assay of LN5P45 with Rho-Ub-PA probe.(A) Gel-based Fluorescence labeling of endogenous OTUB2 in bone-metastatic (BM) MDA-MB-231 cells (left panel) and instant-blue stained loading control (right Panel).BM MDA-MB-231 cells were treated with the indicated concentrations of LN5P45 for 4 hours, followed by cell lysis, incubation with Rho-Ub-PA DUB probe, SDS-PAGE, and gel fluorescence scan.The bands corresponding to OTUB2 are highlighted with red rectangles in the fluorescence scans (B) real-time Quantitative PCR (RT-qPCR) analysis for Otub2 transcripts in BM MDA-MB-231 cells treated with OTUB2 siRNA for 72 hours (Right).Bars represent means ± S.E.M with three samples in each group.Data are representative of four independently performed experiments.

Table S1 .
Overview of the purified recombinant human DUBs used in this study.

Table S2 .
Data processing and refinement statistics for the OTUB2-LN5P45 co-crystal structure.