Structural Dynamics of the Ubiquitin Specific Protease USP30 in Complex with a Cyanopyrrolidine-Containing Covalent Inhibitor

Inhibition of the mitochondrial deubiquitinating (DUB)
enzyme USP30
is neuroprotective and presents therapeutic opportunities for the
treatment of idiopathic Parkinson’s disease and mitophagy-related
disorders. We integrated structural and quantitative proteomics with
biochemical assays to decipher the mode of action of covalent USP30
inhibition by a small-molecule containing a cyanopyrrolidine reactive
group, USP30-I-1. 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 revealed a
slightly different binding mode closer to the active site Cys77, which
may provide the molecular basis for improved selectivity toward USP30
against other members of the DUB enzyme family. Our results highlight
advantages in developing covalent inhibitors, such as USP30-I-1, for targeting USP30 as treatment of disorders with impaired mitophagy.