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.

USP5是一种去泛素化酶，已被证实与包括癌症在内的多种疾病相关，但迄今为止尚未有针对USP5的靶向化学探针被报道。本研究详述了一类化学探针的研发历程：该类探针可结合USP5中特征尚不明确的锌指泛素结合结构域（zinc-finger ubiquitin binding domain，ZnF-UBD）的C端泛素结合位点，并竞争性抑制该酶的催化活性。研究人员通过构效关系探索，并结合ZnF-UBD与多种配体结合的晶体学表征，最终鉴定得到化合物64：其与USP5 ZnF-UBD的解离常数（KD）为2.8 μM，且对9种包含结构相似ZnF-UBD结构域的蛋白具有优异选择性。体外实验结果显示，化合物64可在体外实验中抑制USP5对双泛素底物的催化切割反应。本研究为开发用于阐明细胞内USP5功能的化学探针提供了化学与结构层面的研究框架。