From the NCI-designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037.
From the NCI-designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, California 92037.
J Biol Chem. 2018 Dec 28;293(52):20169-20180. doi: 10.1074/jbc.RA118.004301. Epub 2018 Oct 31.
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.
AAA ATPase p97 调节泛素依赖性蛋白质稳态，已被作为癌症药物靶点进行研究。ATP 竞争性抑制剂 CB-5083 和别构抑制剂 NMS-873 是迄今为止描述的最先进的 p97 抑制剂。以前的研究报告称，它们的细胞毒性很容易被克服，涉及 D1 和 D2 ATP 酶结构域之间的连接区和 D2 内的单个 p97 突变。我们在此报告，位于 D1-D2 连接区的脯氨酸 472 突变为亮氨酸（P472L）突变，在 CB-5083 耐药细胞中被鉴定出来，使 p97 对这两种抑制剂都不敏感。该突变不会破坏抑制剂的不同 D2 结合位点。相反，P472L 改变了 p97 六聚体中 ATP 酶结构域的通讯。P472L 增强了 D2 ATP 的协同结合和水解。这种机制改变了 D1-D2 连接区在控制 D2 活性中的作用，涉及 D1 的 ATP 结合状态。尽管增加的 D2 活性足以使 P472L 突变体对 NMS-873 不敏感，但突变体对 CB-5083 的不敏感也需要 D1 ATP 酶结构域的功能。我们的研究强调了 p97 ATP 酶结构域通讯的惊人适应性，使它能够逃避机制上不同类别的细胞毒性 p97 抑制剂。
