Department of Medical Oncology, Dana-Farber Cancer Institute, 02215, Boston, MA, USA.
Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN, USA.
Cell Death Differ. 2020 Mar;27(3):999-1007. doi: 10.1038/s41418-019-0391-9. Epub 2019 Jul 22.
Inhibition of the anti-apoptotic machinery of cancer cells is a promising therapeutic approach that has driven the development of an important class of compounds termed "BH3 mimetics". These novel small molecules mimic BH3-only proteins by antagonizing the pro-survival function of anti-apoptotic proteins, thereby inducing apoptosis in cancer cells. To qualify as an authentic BH3 mimetic, a compound must function directly on the mitochondria of a cell of known anti-apoptotic dependence, must directly and selectively inhibit the anti-apoptotic protein with high-affinity binding, and must induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis in a BAX/BAK-dependent manner. While many BH3 mimetics have entered clinical trials, the lack of a reliable validation assay to directly test the mitochondrial activity of new BH3 mimetic candidates has resulted in many misleading reports of agents touted as BH3 mimetics despite their off-target mechanisms of action. BH3 profiling probes the activity of a compound at the mitochondrial level by measuring cytochrome c release as a surrogate marker for MOMP. We propose a comprehensive biochemical toolkit consisting of BH3 profiling in parallel with high-throughput Annexin V/Hoechst viability testing to validate BH3 mimetic candidates. We tested our toolkit on eighteen different putative BH3 mimetics using a set of standardized cell lines of known anti-apoptotic dependence. Included in this set of cell lines is an apoptosis refractory BAX/BAK DKO control line to detect compounds that function independently of the BCL-2 family. Taken together, this rapid, efficient means of testing will prove advantageous as the demand for BH3 mimetics increases, particularly in the quest to identify and develop more potent MCL-1 inhibitors for use in the clinic. We strongly urge researchers utilizing BH3 mimetics in their work to use the potent and selective compounds identified with this validation toolkit instead of those lacking such potency and selectivity.
抑制癌细胞的抗凋亡机制是一种很有前途的治疗方法，推动了一类被称为“BH3 模拟物”的重要化合物的发展。这些新型小分子通过拮抗抗凋亡蛋白的生存功能来模拟 BH3 仅蛋白，从而诱导癌细胞凋亡。要成为真正的 BH3 模拟物，一种化合物必须在已知抗凋亡依赖性的细胞的线粒体上直接发挥作用，必须以高亲和力结合直接和选择性地抑制抗凋亡蛋白，并且必须以 BAX/BAK 依赖的方式诱导线粒体膜通透性（MOMP）和凋亡。虽然许多 BH3 模拟物已经进入临床试验，但缺乏可靠的验证测定法来直接测试新的 BH3 模拟物候选物的线粒体活性，导致许多被吹捧为 BH3 模拟物的药物的误导性报告，尽管它们的作用机制存在脱靶。BH3 分析通过测量细胞色素 c 释放作为 MOMP 的替代标志物来探测化合物在线粒体水平的活性。我们提出了一个全面的生化工具包，包括 BH3 分析与高通量 Annexin V/Hoechst 活力测试平行进行，以验证 BH3 模拟物候选物。我们使用一组已知抗凋亡依赖性的标准化细胞系，对十八种不同的假定 BH3 模拟物进行了测试。该细胞系集包括一个凋亡难治性 BAX/BAK DKO 对照系，以检测独立于 BCL-2 家族发挥作用的化合物。总之，这种快速、高效的测试方法将在对 BH3 模拟物的需求增加时证明是有利的，特别是在寻求识别和开发更有效的用于临床的 MCL-1 抑制剂方面。我们强烈敦促在工作中使用 BH3 模拟物的研究人员使用这种验证工具包中鉴定出的有效和选择性化合物，而不是那些缺乏这种效力和选择性的化合物。
