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bioRxiv · 10.1101/2025.10.23.684218

Development of a dual chemical probe for the USP16 and HDAC6 zinc-finger ubiquitin-binding domain

Santhakumar, V.·Silva, M.·Mann, M. K.·Mirabi, B.·Szewczyk, M.·Loup, J.·Dupeux, A.·Chandrasekaran, R.·Bajohr, J.·Arrowsmith, C.·Schapira, M.·Lautens, M.·Barsyte-Lovejoy, D.·Harding, R. J.
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Abstract

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. Because the precise function of USP16-UBD and its interactions remains unclear, a small molecule antagonist targeting the USP16-UBD could enable cellular studies to elucidate its biological role. Here we report SGC-UBD1031 (15), a chemical probe targeting USP16-UBD with similar in vitro binding profiles to HDAC6-UBD and selectivity over nine other UBDs. In cellular assays, 15 disrupts the interaction between the C-terminus of ISG15 and USP1-UBD, as well as the interaction between ISG15 and HDAC6 UBD, at a concentration of 1 M. The corresponding enantiomer SGC-UBD1031N (16), does not interfere with these interactions, even at concentrations as high as 30 M, and thus serves as a negative control. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/684218v2_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@50def6org.highwire.dtl.DTLVardef@15dedc4org.highwire.dtl.DTLVardef@1a50169org.highwire.dtl.DTLVardef@1ffbb4b_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Santhakumar, V., Silva, M., Mann, M. K., Mirabi, B., Szewczyk, M., Loup, J., Dupeux, A., Chandrasekaran, R., Bajohr, J., Arrowsmith, C., Schapira, M., Lautens, M., Barsyte-Lovejoy, D., Harding, R. J.. 2025-10-23. Development of a dual chemical probe for the USP16 and HDAC6 zinc-finger ubiquitin-binding domain. https://doi.org/10.1101/2025.10.23.684218

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