靶向线粒体酪蛋白裂解酶ClpP新型激动剂004A的发现及其体外抗肿瘤活性评价OA

Objective To discover novel agonists targeting human mitochondrial caseinolytic protease P(hClpP).Methods Compounds were screened using an hClpP peptidase activity assay.Target engagement was verified by differential scanning fluorimetry(DSF)and the cellular thermal shift assay(CETSA).Antiprolifera-tive effects on multiple tumor cell lines were assessed by MTT assay,and the effect on tumor cell migration was evaluated by wound healing assay.Cytotoxicity was preliminarily examined in normal hepatocyte LO2 and cardio-myocyte H9C2 cells.Results Compound 004A exhibited a marked agonistic effect on hClpP peptidase activity with an EC50 of 1.31 μmol/L,and concurrently enhanced the degradation of protein substrates casein by hClpP.DSF and CETSA confirmed direct binding of 004A to hClpP.004A inhibited the proliferation of multiple tumor cell lines(IC50 ≈ 10 μmol/L)and suppressed tumor cell migration.Cytotoxicity assays showed that at 50 μmol/L,004A strongly inhibited normal cardiomyocyte H9C2 cells,whereas it exerted only a modest effect on LO2 hepa-tocytes.Conclusion 004A potently activates hClpP,representing a novel,highly effective,and specific hClpP agonist with therapeutic potential against a range of tumor cells,including gastric cancer.Its cardiomyocyte tox-icity,however,indicates that further structural optimization is warranted to improve the safety profile.