VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.
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