Development of a Dual Chemical Probe for the USP16 and HDAC6 Zinc-Finger Ubiquitin-Binding Domain

Ubiquitin-specific peptidase 16 (USP16) is a deubiquitinase that specifically cleaves ubiquitin from histone H2A and modulates gene expression, cell cycle regulation, and various other cellular processes. The USP16 zinc-finger ubiquitin-binding domain (UBD) binds the free C-terminal end of both ubiquitin and ISG15, two major signaling proteins that mediate many biological pathways, but the precise function of USP16-UBD remains unclear. A small-molecule antagonist targeting USP16-UBD could enable cellular studies to elucidate its biological role. Here we report SGC-UBD1031 (<b>15</b>), a chemical probe targeting USP16-UBD with similar <i>in vitro</i> binding profiles to HDAC6-UBD and selectivity over nine other UBDs. In cellular assays, <b>15</b> disrupts the interaction between the C-terminus of ISG15 and USP16-UBD, as well as the interaction between ISG15 and HDAC6-UBD, at a concentration of 1 μM. The corresponding enantiomer SGC-UBD1031N (<b>16</b>) does not interfere with these interactions and thus serves as a negative control.