Recent inclusion of selective DUB inhibitors in clinical trials for the treatment of cancer, kidney and neurodegenerative diseases has heightened interest in DUB-focused drug discovery. We have established a Hydrogen-Deuterium eXchange Mass Spectrometry platform as a tool to capture protein intrinsic disorder in solution [1]. We adapted this approach to profile a panel of DUB – small molecule inhibitor interactions.
We explored the mode of action of covalent USP30 inhibition by a small-molecule containing a cyanopyrrolidine reactive group, USP30-I-1 [2]. The inhibitor demonstrated high potency and selectivity for endogenous USP30 in neuroblastoma cells. Enzyme kinetics and hydrogen-deuterium eXchange mass spectrometry indicated that the inhibitor binds tightly to regions surrounding the USP30 catalytic cysteine and positions itself to form a binding pocket along the thumb and palm domains of the protein, thereby interfering its interaction with ubiquitin substrates. A comparison to a noncovalent USP30 inhibitor containing a benzosulfonamide scaffold [3] revealed a slightly different binding mode closer to the active site Cys77, which may provide the molecular basis for improved potency toward USP30.
We have also applied HDX-MS to determine expanded the repertoire of electrophilic groups targeting DUBs, leading to the discovery of potent and selective inhibitors for USP47, OTUD7B, and USP5 [4]. Using HDX-MS, we reveal the molecular basis of the USP47 inhibitor’s preference over USP7 via selective perturbation of the activation peptide.
Together, we highlight how HDX-MS has been successfully integrated into therapeutic development workflows that can accelerate the development of selective small molecular drug candidates.