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

Discovery of a novel agonist 004A targeting the mitochondrial protease ClpP and evaluation of its antitumor activity in vitro

摘要

目的 靶向人线粒体酪蛋白裂解酶ClpP（hClpP）的新型激动剂研究。方法 通过hClpP肽酶活性检测模型筛选化合物，采用差示扫描荧光法（DSF）和细胞热位移实验（CETSA）验证化合物与靶点的结合。使用MTT法检测化合物对多种肿瘤细胞的增殖抑制效应，通过划痕实验评估其对肿瘤细胞迁移的影响。采用正常肝细胞LO2与心肌细胞H9C2初步评价细胞毒性。结果 化合物004A对hClpP肽酶活性有明显激动作用，EC₅₀为1.31 μmol/L；同时能显著激活hClpP对蛋白底物的降解。DSF和CETSA证实了004A直接结合于hClpP。004A可抑制多种肿瘤细胞的增殖（IC₅₀约10 μmol/L），并抑制肿瘤细胞迁移。细胞毒性实验显示，004A在50 μmol/L下对正常心肌细胞H9C2的抑制较强，而对肝细胞LO2影响较小。结论 004A显著激活hClpP，是一种新型、高效、特异的hClpP激动剂，在包括胃癌在内的多种肿瘤细胞中显示出治疗潜力，尽管其对心肌细胞的毒性提示需进一步结构优化以提高安全性。

Abstract

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). Antiproliferative 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 cardiomyocyte H9C2 cells. Results Compound 004A exhibited a marked agonistic effect on hClpP peptidase activity with an EC ₅₀ 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 (IC ₅₀≈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 hepatocytes. 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 toxicity, however, indicates that further structural optimization is warranted to improve the safety profile.

关键词

线粒体 / 酪蛋白裂解酶 / 蛋白质稳态 / 激动剂 / 抗肿瘤活性

Key words

mitochondria / ClpP / protein homeostasis / agonist / antitumor activity

参考文献

[1] Wang C, Youle RJ. The role of mitochondria in apoptosis[J]. Annu Rev Genet, 2009, 43: 95-118.

[2] Osellame LD, Blacker TS, Duchen MR. Cellular and molecular mechanisms of mitochondrial function[J]. Best Pract Res Clin Endocrinol Metab, 2012, 26(6): 711-723.

[3] Ward PS, Thompson CB. Metabolic reprogramming: a cancer hallmark even warburg did not anticipate[J]. Cancer Cell, 2012, 21(3): 297-308.

[4] Du H, Xu T, Yu S, et al. Mitochondrial metabolism and cancer therapeutic innovation[J]. Signal Transduct Target Ther, 2025, 10(1): 245.

[5] Sies H, Jones DP. Reactive oxygen species (ROS) as pleiotropic physiological signalling agents[J]. Nat Rev Mol Cell Biol, 2020, 21(7): 363-383.

[6] Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach?[J]. Nat Rev Drug Discov, 2009, 8(7): 579-591.

[7] Cheung EC, Vousden KH. The role of ROS in tumour development and progression[J]. Nat Rev Cancer, 2022, 22(5): 280-297.

[8] Kenny TC, Gomez ML, Germain D. Mitohormesis, UPR(mt), and the complexity of mitochondrial DNA landscapes in cancer[J]. Cancer Res, 2019, 79(24): 6057-6066.

[9] Costa-Mattioli M, Walter P. The integrated stress response: from mechanism to disease[J]. Science, 2020, 368(6489): eaat5314.

[10] Song J, Herrmann JM, Becker T. Quality control of the mitochondrial proteome[J]. Nat Rev Mol Cell Biol, 2021, 22(1): 54-70.

[11] Graves PR, Aponte-Collazo LJ, Fennell EMJ, et al. Mitochondrial protease ClpP is a target for the anticancer compounds ONC201 and related analogues[J]. ACS Chem Biol, 2019, 14(5): 1020-1029.

[12] Zhang Y, Maurizi MR. Mitochondrial ClpP activity is required for cisplatin resistance in human cells[J]. Biochim Biophys Acta Mol Basis Dis, 2016, 1862(2): 252-264.

[13] Armenise D, Baldelli OM, Liturri A, et al. Mitochondrial protease ClpP: cancer marker and drug target[J]. Pharmaceuticals (Basel), 2025, 18(10): 1443.

[14] Shu Z, Zhang J, Wu Y, et al. Recent advances in the design of small molecules targeting human ClpP[J]. Future Med Chem, 2025, 17(19): 2407-2424.

[15] Liu X, Gu J, Liu S, et al. The mitochondrial protease ClpP is a promising target for multiple myeloma treatment[J]. Biochem Pharmacol, 2025, 236: 116855.

[16] Chattopadhyay C, Roszik J, Bhattacharya R, et al. Imipridones inhibit tumor growth and improve survival in an orthotopic liver metastasis mouse model of human uveal melanoma[J]. Br J Cancer, 2024, 131(11): 1846-1857.

[17] Nandha SR, Patwardhan RS, Sharma D, et al. Targeting mitochondrial proteases CLPP and LONP1 via disruption of mitochondrial redox homeostasis induces proteotoxic stress and suppresses tumor progression[J]. Cell Commun Signal, 2025, 23(1): 393.

[18] Nouri K, Feng Y, Schimmer AD. Mitochondrial ClpP serine protease-biological function and emerging target for cancer therapy[J]. Cell Death Dis, 2020, 11(10): 841.

[19] Seo JH, Rivadeneyra DB, Caino MC, et al. The Mitochondrial unfoldase-peptidase complex clpxp controls bioenergetics stress and metastasis[J]. PLoS Biol, 2016, 14(7): e1002507.

[20] Cole A, Wang Z, Coyaud E, et al. Inhibition of the mitochondrial protease ClpP as a therapeutic strategy for human acute myeloid leukemia[J]. Cancer Cell, 2015, 27(6): 864-876.

[21] Culp E, Wright GD. Bacterial proteases, untapped antimicrobial drug targets[J]. J Antibiot (Tokyo), 2017, 70(4): 366-377.

[22] Alexopoulos JA, Guarne A, Ortega J. ClpP: a structurally dynamic protease regulated by AAA+ proteins[J]. J Struct Biol, 2012, 179(2): 202-210.

[23] Bonner ER, Waszak SM, Grotzer MA, et al. Mechanisms of imipridones in targeting mitochondrial metabolism in cancer cells[J]. Neuro Oncol, 2021, 23(4): 542-556.

[24] Xiang X, Dai Z, Luo B, et al. Rational design of a novel class of human ClpP agonists through a ring-opening strategy with enhanced antileukemia activity[J]. J Med Chem, 2024, 67(8): 6769-6792.

[25] Huang J, Zhang J, Luo B, et al. Discovery of a novel series of imipridone compounds as homo sapiens caseinolytic protease p agonists with potent antitumor activities in vitro and in vivo[J]. J Med Chem, 2022, 65(11): 7629-7655.

[26] Zhang J, Qiu Z, Liu S, et al. Discovery of a novel series of homo sapiens caseinolytic protease P agonists for colorectal adenocarcinoma treatment via ATF3-dependent integrated stress response[J]. J Med Chem, 2024, 67(4): 2812-2836.

[27] Xu Z, Pokushalov D, Kabir M, et al. Targeting the mitochondrial protease ClpP for anticancer therapy[J]. J Med Chem, 2025, 68(20): 21377-21393.

[28] Zhang J, Luo B, Sui J, et al. IMP075 targeting ClpP for colon cancer therapy in vivo and in vitro[J]. Biochem Pharmacol, 2022, 204: 115232.

[29] Wart SV, Phillips L, Ludwig EA, et al. Population pharmacokinetics and pharmacodynamics of garenoxacin in patients with community-acquired respiratory tract infections[J]. Antimicrob Agents Chemother, 2004, 48(12): 4766-4777.

[30] Wong KS, Mabanglo MF, Seraphim TV, et al. Acyldepsipeptide analogs dysregulate human mitochondrial ClpP protease activity and cause apoptotic cell death[J]. Cell Chem Biol, 2018, 25(8): 1017-1030.

[31] Ishizawa J, Zarabi SF, Davis RE, et al. Mitochondrial ClpP-Mediated proteolysis induces selective cancer cell lethality[J]. Cancer Cell, 2019, 35(5): 721-737.

[32] Nii T, Prabhu VV, Ruvolo V, et al. Imipridone ONC212 activates orphan G protein-coupled receptor GPR132 and integrated stress response in acute myeloid leukemia[J]. Leukemia, 2019, 33(12): 2805-2816.

[33] Pearson AD, Mueller S, Filbin MG, et al. Paediatric strategy for umformed medicinal product development in diffuse midline gliomas in children and adolescents ACCEL-ERATE in collaboration with the European Medicines Agency with participation of the Food and Drug Administration[J]. Eur J Cancer, 2025, 217: 115230.

[34] Wong KS, Houry WA. Chemical modulation of human mitochondrial ClpP: potential application in cancer therapeutics[J]. ACS Chem Biol, 2019, 14(11): 2349-2360.

[35] Jackson ER, Duchatel RJ, Staudt DE, et al. ONC201 in combination with paxalisib for the treatment of H3K27-altered diffuse midline glioma[J]. Cancer Res, 2023: Of1-Of17.

[36] Miciaccia M, Baldelli OM, Fortuna CG, et al. ONC201-derived tetrahydropyridopyrimidindiones as powerful ClpP protease activators to tackle diffuse midline glioma[J]. J Med Chem, 2025, 68(5): 5190-5210.

[37] Odia Y, Hall MD, Cloughesy TF, et al. Selective DRD2 antagonist and ClpP agonist ONC201 in a recurrent non-midline H3K27M-mutant glioma cohort[J]. Neuro Oncol, 2024, 26(Supplement_2): S165-S172.

[38] Sathe S, Li Q, Jung J, et al. Targeting the mitochondria in high-grade gliomas[J]. Cancers (Basel), 2025, 17(18): 3062.

[39] Kim YI, Levchenko I, Fraczkowska K, et al. Molecular determinants of complex formation between Clp/Hsp100 ATPases and the ClpP peptidase[J]. Nat Struct Biol, 2001, 8(3): 230-233.

[40] Akopian T, Kandror O, Raju RM, et al. The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring[J]. EMBO J, 2012, 31(6): 1529-1541.

[41] Vollrath F, Hawkins N, Porter D, et al. Differential scanning fluorimetry provides high throughput data on silk protein transitions[J]. Sci Rep, 2014, 4: 5625.

[42] Jafari R, Almqvist H, Axelsson H, et al. The cellular thermal shift assay for evaluating drug target interactions in cells[J]. Nat Protoc, 2014, 9(9): 2100-2122.

[43] Prabhu VV, Allen JE, Dicker DT, et al. Small-molecule ONC201/TIC10 targets chemotherapy-resistant colorectal cancer stem-like cells in an Akt/Foxo3a/TRAIL-dependent manner[J]. Cancer Res, 2015, 75(7): 1423-1432.

[44] Parker CS, Zhou L, Prabhu VV, et al. ONC201/TIC10 plus TLY012 anti-cancer effects via apoptosis inhibitor downregulation, stimulation of integrated stress response and death receptor DR5 in gastric adenocarcinoma[J]. Am J Cancer Res, 2023, 13(12): 6290-6312.

[45] Cao YP, Zheng M. Mitochondrial dynamics and intermitochondrial communication in the heart[J]. Arch Biochem Biophys, 2019, 663: 214-219.

[46] Gao J, Shen W. Sirtuin-3-mediated cellular metabolism links cardiovascular remodeling with hypertension[J]. Biology (Basel), 2023, 12(5): 686.

[47] Houten SM, Violante S, Ventura FV, et al. The biochemistry and physiology of mitochondrial fatty acid β-oxidation and its genetic disorders[J]. Annu Rev Physiol, 2016, 78: 23-44.

[48] Tannous P, Zhu H, Johnstone JL, et al. Autophagy is an adaptive response in desmin-related cardiomyopathy[J]. Proc Natl Acad Sci USA, 2008, 105(28): 9745-9750.

[49] Smith RA, Hartley RC, Murphy MP. Mitochondria-targeted small molecule therapeutics and probes[J]. Antioxid Redox Signal, 2011, 15(12): 3021-3038.

[50] Siegel RL, Miller KD, Wagle NS, et al. Cancer statistics, 2023[J]. CA Cancer J Clin, 2023, 73(1): 17-48.

[51] Sundar R, Nakayama I, Markar SR, et al. Gastric cancer[J]. Lancet, 2025, 405(10494): 2087-2102.

[52] Luo D, Liu Y, Lu Z, et al. Targeted therapy and immunotherapy for gastric cancer: rational strategies, novel advancements, challenges, and future perspectives[J]. Mol Med, 2025, 31(1): 52.

[53] Wang G, Huang Y, Zhou L, et al. Immunotherapy and targeted therapy as first-line treatment for advanced gastric cancer[J]. Crit Rev Oncol Hematol, 2024, 198: 104197.

[54] Huang L, Liu H, Yu J, et al. Long-term outcomes in laparoscopic D2 gastrectomy for gastric cancer: a large comprehensive study proposing novel hypotheses[J]. J Gastrointest Surg, 2019, 23(7): 1349-1361.

[55] Huang L, Yang H, Chen K, et al. The suppressive efficacy of THZ1 depends on KRAS mutation subtype and is associated with super-enhancer activity and the PI3K/AKT/mTOR signalling in pancreatic ductal adenocarcinoma: a hypothesis-generating study[J]. Clin Transl Med, 2023, 13(12): e1500.

[56] Chen ZM, Huang L, Li MM, et al. Inhibitory effects of isocryptotanshinone on gastric cancer[J]. Sci Rep, 2018, 8(1): 9307.

[57] Smyth EC, Nilsson M, Grabsch HI, et al. Gastric cancer[J]. Lancet, 2020, 396(10251): 635-648.

基金资助

北京市自然科学基金面上项目(7252193)