Discovery and Characterization of a Chemical Probe Targeting the Zinc-Finger Ubiquitin-Binding Domain of HDAC6

Histone deacetylase 6 (HDAC6) inhibition is an attractivestrategyfor treating numerous cancers, and HDAC6 catalytic inhibitors arecurrently in clinical trials. The HDAC6 zinc-finger ubiquitin-bindingdomain (UBD) binds free C-terminal diglycine motifs of unanchoredubiquitin polymer chains and protein aggregates, playing an importantrole in autophagy and aggresome assembly. However, targeting thisdomain with small molecule antagonists remains an underdeveloped avenueof HDAC6-focused drug discovery. We report SGC-UBD253 (25), a chemical probe potently targeting HDAC6-UBDin vitro with selectivity over nine other UBDs, except for weak USP16binding. In cells, 25 is an effective antagonist of HDAC6-UBDat 1 & mu;M, with marked proteome-wide selectivity. We identified SGC-UBD253N (32), a methylated derivative of 25 that is 300-fold less active, serving as a negative control.Together, 25 and 32 could enable furtherexploration of the biological function of the HDAC6-UBD and investigationof the therapeutic potential of targeting this domain.