The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs， such as Navitoclax and Venetoclax， in treating BCL-2-dependent haematological malignancies. However， most solid tumours depend on MCL-1 for survival， which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far， while demonstrating early promise in vitro， fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects， we developed a rapid differential scann... More
The concept of using BH3 mimetics as anticancer agents has been substantiated by the efficacy of selective drugs， such as Navitoclax and Venetoclax， in treating BCL-2-dependent haematological malignancies. However， most solid tumours depend on MCL-1 for survival， which is highly amplified in multiple cancers and a major factor determining chemoresistance. Most MCL-1 inhibitors that have been generated so far， while demonstrating early promise in vitro， fail to exhibit specificity and potency in a cellular context. To address the lack of standardised assays for benchmarking the in vitro binding of putative inhibitors before analysis of their cellular effects， we developed a rapid differential scanning fluorimetry (DSF)-based assay， and used it to screen a panel of BH3 mimetics. We next contrasted their binding signatures with their ability to induce apoptosis in a MCL-1 dependent cell line. Of all the MCL-1 inhibitors tested， only A-1210477 induced rapid， concentration-dependent apoptosis， which strongly correlated with a thermal protective effect on MCL-1 in the DSF assay. In cells that depend on both MCL-1 and BCL-X， A-1210477 exhibited marked synergy with A-1331852， a BCL-X specific inhibitor， to induce cell death. Despite this selectivity and potency， A-1210477 induced profound structural changes in the mitochondrial network in several cell lines that were not phenocopied following MCL-1 RNA interference or transcriptional repression， suggesting that A-1210477 induces mitochondrial fragmentation in an MCL-1-independent manner. However， A-1210477-induced mitochondrial fragmentation was dependent upon DRP-1， and silencing expression levels of DRP-1 diminished not just mitochondrial fragmentation but also BH3 mimetic-mediated apoptosis. These findings provide new insights into MCL-1 ligands， and the interplay between DRP-1 and the anti-apoptotic BCL-2 family members in the regulation of apoptosis.
