ABSTRACT
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 allosterically inhibits the catalytic activity of the enzyme. Systematic 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. 64 is selective over the structurally similar ZnF-UBD domain of HDAC6 and inhibits USP5 catalytic activity in vitro with an IC50 of 26 µM. This study provides a chemical and structural framework for the discovery of a chemical probe to delineate USP5 function in cells.
Competing Interest Statement
The authors have declared no competing interest.
ABBREVIATIONS
- BRAP
- BRCA-1 associated protein
- DCM
- dichloromethane
- DMF
- dimethylformamide
- DMSO
- dimethylsulfoxide
- DIPEA
- N,N-diisopropylethylamine
- DUB
- deubiquitinase
- FL
- full-length
- HATU
- hexafluorophosphate azabenzotriazole tetramethyl uranium
- HDAC6
- histone deacetylase 6
- HRMS
- high resolution mass spectrometry
- IQF
- internally quenched fluorophore
- KD
- dissociation constant
- LCMS
- liquid chromatography mass spectrometry
- LRMS
- low resolution mass spectrometry
- QTOF
- quadrupole-time-of-flight
- SAR
- structure activity relationship
- SD
- standard deviation
- SPE
- solid phase extraction
- SPR
- surface plasmon resonance
- TFA
- trifluoroacetic acid
- Ub
- ubiquitin
- Ub2K48
- Lys48-linked di-ubiquitin
- UBA
- ubiquitin-binding associated domain
- Ub-AMC
- ubiquitin amidomethyl coumarin
- nUBP
- N-terminal ubiquitin-binding domain
- USP
- ubiquitin specific protease
- UPLC
- ultra-performance liquid chromatography
- WT
- wild-type
- ZnF-UBD
- zinc finger ubiquitin-binding domain