Zhang Jiangnan, Luo Baozhu, Sui Jing, Qiu Zhiqiang, Huang Jiasheng, Yang Tao, Luo Youfu
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China.
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, China; Institute of Immunology and Inflammation, Frontiers Science Center for Disease-related Molecular Network, West China Hospital, Sichuan University, Chengdu 610041, China.
Biochem Pharmacol. 2022 Oct;204:115232. doi: 10.1016/j.bcp.2022.115232. Epub 2022 Aug 27.
ONC201 is a well-known caseinolytic protease (ClpP)activator with established benefits against multiple tumors, including colorectal cancer (CRC). In this study, we investigated the anticancer effects and associated mechanisms of the ClpP agonist IMP075, derived from ONC201. Acute toxicity and CCK-8 assayswere employed to determine the safety of IMP075. The effectiveness of IMP075 was investigated in HCT116 cells and a mouse xenograft tumor model. Additionally, the properties of IMP075 were evaluated by pharmacokinetic,CYP inhibition, and hERG inhibition assays. Finally, isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF), cellular thermal shift assay (CETSA), molecular dynamics simulations, point mutations, and shRNA experiments were employed to elucidate the potential mechanism of IMP075. Compared with ONC201, IMP075 exhibited similar toxicity and improved antitumor effects in vitro and in vivo. Interestingly, the affinity and agonistic effects of IMP075 on ClpP were superior to ONC201, which allowed IMP075 to disrupt respiratory chain integrity at lower doses in HCT116 cells, leading to mitochondrial dysfunction. Furthermore, molecular dynamics simulations demonstrate that IMP075 forms two pairs of hydrogen bonds with ClpP, maintaining ClpP in an agonistic state. Importantly, the antiproliferative activity of IMP075 significantly decreased following ClpP knockdown. Our findings substantiate that IMP075 exerts excellent antitumor effects against CRC by activating ClpP-mediated impairment of mitochondrial function. Due to its superior properties, IMP075 appears to be have huge prospects for application.
ONC201是一种著名的酪蛋白溶解蛋白酶（ClpP）激活剂，对包括结直肠癌（CRC）在内的多种肿瘤具有既定的益处。在本研究中，我们研究了源自ONC201的ClpP激动剂IMP075的抗癌作用及相关机制。采用急性毒性和CCK - 8试验来确定IMP075的安全性。在HCT116细胞和小鼠异种移植肿瘤模型中研究了IMP075的有效性。此外，通过药代动力学、CYP抑制和hERG抑制试验评估了IMP075的特性。最后，采用等温滴定量热法（ITC）、差示扫描荧光法（DSF）、细胞热位移分析（CETSA）、分子动力学模拟、点突变和shRNA实验来阐明IMP075的潜在机制。与ONC201相比，IMP075在体外和体内表现出相似的毒性并具有更好的抗肿瘤作用。有趣的是，IMP075对ClpP的亲和力和激动作用优于ONC201，这使得IMP075能够在较低剂量下破坏HCT116细胞中的呼吸链完整性，导致线粒体功能障碍。此外，分子动力学模拟表明IMP075与ClpP形成两对氢键，使ClpP保持激动状态。重要的是，ClpP敲低后IMP075的抗增殖活性显著降低。我们的研究结果证实，IMP075通过激活ClpP介导的线粒体功能损伤对CRC发挥出色的抗肿瘤作用。由于其优越的特性，IMP075似乎具有巨大的应用前景。
