Structural basis for the bi-specificity of USP25 and USP28 inhibitors

The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets. The inhibitors AZ1, VSM and FT206 bind USP28 and USP25 in the same conserved cleft which explains their bi-specificity. A glutamate only found in both homologs, plays a central role for inhibition through multiple mechanisms.AZ1, VSM and FT206 bind in an extended cleft between the thumb and palm subdomains. Inhibition is achieved by a common allosteric mechanism. The poorly conserved USP28 E366 and USP25 E373 residues impair catalytic activity and promote inhibitor binding. The inhibitors AZ1, VSM and FT206 bind USP28 and USP25 in the same conserved cleft which explains their bi-specificity. A glutamate only found in both homologs, plays a central role for inhibition through multiple mechanisms.