SHIH-HSUN CHENWu, Szu-YingSzu-YingWuChang, Yun-XunYun-XunChangLui, En-NingEn-NingLuiChang, Chih-KangChih-KangChangLin, Sheng-WeiSheng-WeiLinHsiao, MichaelMichaelHsiaoYang, Jinn-MoonJinn-MoonYangLiang, Po-HuangPo-HuangLiang2025-07-182025-07-182025-06-18https://www.scopus.com/record/display.uri?eid=2-s2.0-105008700456&origin=resultslisthttps://scholars.lib.ntu.edu.tw/handle/123456789/730737X-linked inhibitor of apoptosis (XIAP) inhibits caspases 3, 7, and 9, thereby preventing cell apoptosis. Endogenous Second mitochondria-derived activator of caspase (Smac) competes out the binding of caspases with XIAP and causes apoptosis, so that Smac mimetics are under clinical trials for anti-cancer chemotherapy. We demonstrated by selectively alkylating caspase 7 (CASP7) to release the active CASP7 for killing the drug-resistant cancer cells with accumulated XIAP:CASP7 resulted from caspase-3 down-regulation (CASP3/DR). However, finding a reversible inhibitor of the protein-protein interaction (PPI) poses a significant challenge. Here, we identified a reversible XIAP:CASP7 inhibitor, 643943, through a multiple-mode virtual screening strategy. In vitro experiments revealed that 643943 bound to CASP7, released the linker-BIR2 domain of XIAP, and activated the caspase. Removing an essential hydroxyl group on 643943 or replacing the OH-interacting Asp93 on CASP7 caused loss of 643943 cytotoxicity, revealing the binding mode. This compound thus selectively killed MCF-7 and other CASP3/DR triple-negative breast cancer cell lines, but not the cancer and normal cell lines expressing higher levels of CASP3 in vitro and in vivo. Moreover, 643943 overcame chemoresistance via down-regulating β-catenin and its associated ABC transporters in paclitaxel-resistant MCF-7 cells. Our studies not only serve as a proof-of-concept for using XIAP:CASP7 as a drug target, but also provide the first reversible XIAP:CASP7 inhibitor for cancer therapy of CASP3/DR malignanciesen[SDGs]SDG3journal article10.1038/s41419-025-07774-y