Fig. 1. Biochemical and cellular characterization of compounds MT16-001, MT16-205 and MT16-009. (a) Mechanism of cyanamide warhead labeling of a reactive cysteine residue. (b) Structures of compounds MT16-001, MT16-205 and MT16-009 and their activities in biochemical enzymatic, cellular cytotoxicity and target engagement assays. (c) Biochemical selectivity profiling of compounds MT16-001, MT16-205 and MT16-009 at 1 μM using fluorescence polarization (FP) and fluorescence intensity (FI) in vitro assays. (d and e) cellular target engagement of MT16-205 using HA-Ub-VME ABP analyzed by immunoblotting against (d) HA antibody for whole DUB inhibition profiling or (e) DUB antibodies for selectivity profiling. HSP90 was used as a protein loading control.