Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogues
Mitochondrial Protease ClpP is a Target for the Anticancer Compounds ONC201 and Related Analogues
复制标题
DOI：
10.1021/acschembio.9b00222
复制
发表时间：
2019-05-01
影响因子：
4
通讯作者：
Graves, Lee M.
中科院分区：
文献类型：
--
作者：
Graves, Paul R.;Aponte-Collazo, Lucas J.;Graves, Lee M.
ONC 201是目前用于治疗多种癌症的临床试验中的一流的依米立酮分子。尽管具有巨大的临床潜力，但其作用机制仍存在争议。为了研究ONC 201的机制并鉴定具有改善效力的化合物，我们测试了一系列新型ONC 201类似物（TR化合物）对乳腺癌和其他癌症模型中细胞活力和应激反应的影响。发现TR化合物在抑制细胞增殖和诱导整合的应激反应蛋白ATF 4方面比ONC 201强50-100倍。使用固定化TR化合物，我们通过质谱鉴定了人线粒体酪蛋白分解蛋白酶P（CipP）作为特异性结合蛋白。亲和层析/药物竞争测定显示，TR化合物以与ONC 201相比高10倍的亲和力结合ClpP。重要的是，我们发现重组ClpP的肽酶活性被ONC 201和TR化合物以剂量和时间依赖性方式强烈激活，TR化合物显示出与ONC 201相比类似的10-100倍的效力增加。最后，SUM 159细胞中ClpP的siRNA敲低降低了对ONC 201和TR化合物的应答，包括CHOP的诱导、线粒体蛋白（TEAM、TUFM）的损失以及这些化合物的细胞抑制作用。因此，我们报道ClpP直接结合ONC 201和相关的TR化合物，并且是这类分子的重要生物靶标。此外，这些研究首次为ONC 201和TR化合物之间的功效差异提供了生物化学基础。
ONC201 is a first-in-class imipridone molecule currently in clinical trials for the treatment of multiple cancers. Despite enormous clinical potential, the mechanism of action is controversial. To investigate the mechanism of ONC201 and identify compounds with improved potency, we tested a series of novel ONC201 analogues (TR compounds) for effects on cell viability and stress responses in breast and other cancer models. The TR compounds were found to be similar to 50-100 times more potent at inhibiting cell proliferation and inducing the integrated stress response protein ATF4 than ONC201. Using immobilized TR compounds, we identified the human mitochondrial caseinolytic protease P (CIpP) as a specific binding protein by mass spectrometry. Affinity chromatography/drug competition assays showed that the TR compounds bound CIpP with similar to 10-fold higher affinity compared to ONC201. Importantly, we found that the peptidase activity of recombinant ClpP was strongly activated by ONC201 and the TR compounds in a dose- and time-dependent manner with the TR compounds displaying a similar to 10-100 fold increase in potency over ONC201. Finally, siRNA knockdown of ClpP in SUM159 cells reduced the response to ONC201 and the TR compounds, including induction of CHOP, loss of the mitochondrial proteins (TEAM, TUFM), and the cytostatic effects of these compounds. Thus, we report that ClpP directly binds ONC201 and the related TR compounds and is an important biological target for this class of molecules. Moreover, these studies provide, for the first time, a biochemical basis for the difference in efficacy between ONC201 and the TR compounds.