Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors

The AAA⁺ ATPase p97 regulates ubiquitin-dependent protein homeostasis and has been pursued as a cancer drug target. The ATP-competitive inhibitor CB-5083 and allosteric inhibitor NMS-873 are the most advanced p97 inhibitors described to date. Previous studies have reported that their cytotoxicity can be readily overcome and involves single p97 mutations in the linker between the D1 and D2 ATPase domains and within D2. We report here that the proline 472 to leucine (P472L) mutation, in the D1-D2 linker and identified in CB-5083-resistant cells, desensitizes p97 to both inhibitor classes. This mutation does not disrupt the distinct D2-binding sites of the inhibitors. Instead, P472L changes ATPase domain communication within the p97 hexamer. P472L enhances cooperative D2 ATP binding and hydrolysis. This mechanism alters the function of the D1-D2 linker in the control of D2 activity involving the ATP-bound state of D1. Although increased D2 activity is sufficient to desensitize the P472L mutant to NMS-873, the mutant's desensitization to CB-5083 also requires D1 ATPase domain function. Our study highlights the remarkable adaptability of p97 ATPase domain communication that enables escape from mechanistically distinct classes of cytotoxic p97 inhibitors.