表達純化全長 XIAP來檢測 XIAP:CASP7 的可逆抑制劑

Abstract

X-linked inhibitor of apoptosis (XIAP) inhibits caspases 3 (CASP3), 7 (CASP7), and 9, thereby preventing cell apoptosis. XIAP overexpression has been found in chemoresistant cancer cells. Therefore, several small-molecule XIAP antagonists and SMAC mimetics have been developed to inhibit the protein-protein interaction between XIAP and these caspases to kill tumor cells. XIAP utilizes the second repeat (BIR2) domain and the linker between BIR1 and BIR2 to bind and inhibit CASP3/7 in the intrinsic apoptotic pathway. Due to a lack of selectivity between CASP3 and 7 by targeting XIAP, some of these compounds might be toxic to cells. We previously reported a strategy by selectively targeting CASP7 to disrupt XIAP:CASP7 complex but not XIAP:CASP3, so that the compound killed CASP3-downregulated (CASP3/DR) cancer cells resulted from chemotherapy, which have accumulated XIAP:CASP7. A reversible XIAP:CASP7 inhibitor identified by Dr. Shih-Hsun Chen through a multiple-mode virtual screening strategy was found to bind CASP7, release the linker-BIR2 domain of XIAP, and activate CASP7 for killing CASP3/DR cancers cells (Chen et al., unpublished). I therefore aimed to express and purify the full-length XIAP for confirming that the compound can disrupt the interaction between the full-length XIAP and CASP7. I demonstrated that the compound can disrupt the interaction of the full-length XIAP with CASP7 by using CASP7 activity assay, fluorescence quenching, and BIAcore experiments.