ZK53

ZK53 (CAS No. 3031789-26-8) is a highly selective, potent activator of human mitochondrial serine protease ClpP (HsClpP), with no activating effect on bacterial ClpP from Staphylococcus aureus, Escherichia coli, Lactobacillus reuteri, and others. Its EC₅₀ for HsClpP is 0.22μM by the FI assay and 1.37μM by the PAGE assay, and it can increase the melting temperature of HsClpP by 16.1℃. Its MIC for gut probiotics is ＞128μg/mL, and its GI₅₀ for lung squamous cell carcinoma H1703 cells is 0.55μM; this compound activates two core biological pathways mediated by HsClpP: first, it degrades mitochondrial electron transport chain subunits and disrupts oxidative phosphorylation, thereby activating the ATM-mediated DNA damage response and inhibiting E2F target genes, triggering G0/G1 cell-cycle arrest and apoptosis in tumor cells; second, it increases mitochondrial ROS via ClpP-dependent mitochondrial dysfunction, enhancing tumor cell sensitivity to ferroptosis inducers and amplifying lipid peroxidation.

In cell experiments, commonly used non-toxic working concentrations are 10μM for HT-1080 cells, 1μM for HeLa cells, and 5μM for HCT-116 cells; effective anti-proliferative and ferroptosis-sensitizing concentrations range from nanomolar to low micromolar levels.

In animal experiments, it has been used for treatment in a lung squamous cell carcinoma xenograft nude mouse model, a spontaneous lung squamous cell carcinoma KL mouse model, and an HCT-116 colorectal cancer nude mouse model. In the lung squamous cell carcinoma model, the intraperitoneal dose is 80mg/kg (twice daily); in the colorectal cancer model combined with the ferroptosis inducer IKE, the intraperitoneal dose is 20mg/kg (once every other day), with no obvious organ toxicity or significant body-weight loss.

References:

[1] Zhou LL, Zhang T, Xue Y, Yue C, Pan Y, Wang P, Yang T, Li M, Zhou H, Ding K, Gan J, Ji H, Yang CG. Selective activator of human ClpP triggers cell cycle arrest to inhibit lung squamous cell carcinoma. Nat Commun. 2023 Nov 3;14(1):7069. doi: 10.1038/s41467-023-42784-4. PMID: 37923710; PMCID: PMC10624687.

[2] Shu Z, Zhang J, Wu Y, Yang T, Luo Y. Recent advances in the design of small molecules targeting human ClpP. Future Med Chem. 2025 Oct;17(19):2407-2424. doi: 10.1080/17568919.2025.2557175. Epub 2025 Sep 8. PMID: 40916604; PMCID: PMC12492974.

[3] Dai S, Zhang T, Dai X, Zheng K, Qian H, Tong Z, Zhang Q. ZK53 enhances tumor cell susceptibility to ferroptosis via ClpP-mediated mitochondrial dysfunction. Front Oncol. 2025 Nov 21;15:1594840. doi: 10.3389/fonc.2025.1594840. PMID: 41357605; PMCID: PMC12678104.