Re-Evaluating the Mechanism of Action of α,β-Unsaturated Carbonyl DUB Inhibitors b‑AP15 and VLX1570: A Paradigmatic Example of Unspecific Protein Cross-linking with Michael Acceptor Motif-Containing Drugs

Deubiquitinating
enzymes (DUBs) are a growing target class across
multiple disease states, with several inhibitors now reported. b-AP15
and VLX1570 are two structurally related USP14/UCH-37 inhibitors.
Through a proteomic approach, we demonstrate that these compounds
target a diverse range of proteins, resulting in the formation of
higher molecular weight (MW) complexes. Activity-based proteome profiling
identified CIAPIN1 as a submicromolar covalent target of VLX1570,
and further analysis demonstrated that high MW complex formation leads
to aggregation of CIAPIN1 in intact cells. Our results suggest that
in addition to DUB inhibition, these compounds induce nonspecific
protein aggregation, providing molecular explanation for general cellular
toxicity.