Structure–Activity Relationship of USP5 Inhibitors

USP5
is a deubiquitinase that has been implicated in a range of
diseases, including cancer, but no USP5-targeting chemical probe has
been reported to date. Here, we present the progression of a chemical
series that occupies the C-terminal ubiquitin-binding site of a poorly
characterized zinc-finger ubiquitin binding domain (ZnF-UBD) of USP5
and competitively inhibits the catalytic activity of the enzyme. Exploration
of the structure–activity relationship, complemented with crystallographic
characterization of the ZnF-UBD bound to multiple ligands, led to
the identification of 64, which binds to the USP5 ZnF-UBD
with a KD of 2.8 μM and is selective
over nine proteins containing structurally similar ZnF-UBD domains. 64 inhibits the USP5 catalytic cleavage of a di-ubiquitin
substrate in an in vitro assay. This study provides
a chemical and structural framework for the discovery of a chemical
probe to delineate USP5 function in cells.