Small Molecule Activators of Pro-apoptotic BAX for Cancer Therapy

摘要 对细胞凋亡的抗性是癌症的标志之一。癌细胞绕过细胞死亡，通过 线粒体凋亡途径，以确保肿瘤生长，维持和抵抗目前的治疗。 线粒体凋亡途径由BCL-2家族的表达水平和相互作用控制 蛋白，其包括促凋亡效应蛋白BAX和巴克，抗凋亡蛋白例如BCL- 2、BCL-XL、MCL-1和促凋亡蛋白BH 3-only。癌细胞最常阻断线粒体 通过上调抗凋亡BCL-2蛋白来中和仅BH 3蛋白和激活BAX 还有巴克。因此，努力集中在抗凋亡BCL-2的选择性抑制剂的开发上 蛋白质来重新激活凋亡。抑制剂，如FDA批准的维奈托克依赖于功能， BH 3-only蛋白激活BAX和巴克的可用性。然而，在许多肿瘤中，BH 3-only蛋白质可以是 下调、抑制或删除，使这些肿瘤对这些抑制剂不敏感，并限制其 更广泛的临床应用。我们假设，小分子直接激活促凋亡BAX通过 BAX触发位点是一种替代的和可能的补充药理学策略，以促进细胞凋亡， 癌细胞这种方法可以促进BAX激活独立于BH 3-only蛋白，因此 应该具有克服耐药肿瘤中的细胞凋亡阻断的潜力。我们的实验室最近使用了 独特的结构和分子见解和药物化学，以开发一种有效的和选择性的化合物， 称为BAX触发位点激活物1（BTSA 1），其促进BAX激活并诱导线粒体 功能障碍和凋亡。使用BTSA 1，我们提供了直接BAX激活作为治疗方法的概念验证。 靶向急性髓性白血病，并证明直接BAX活化在体内耐受良好。这里我们 假设产生具有改善的效力、口服生物利用度和药代动力学的BTSA 1类似物。 我们的目标是在不同的癌症模型中评估它们作为单一药物或 联合治疗，并研究对BAX活化的敏感性和抗性机制， 凋亡此外，我们的目标是鉴定具有有利的细胞， 药理学和安全性。因此，我们提出以下具体目标：1）表征 BTSA 1的效力、选择性、促凋亡活性、体外ADME/Tox和药代动力学特性 类似物，2）确定第二代BTSA，包括BTSA 1.2， 使用各种白血病和实体瘤细胞进行单独和联合治疗，并研究决定因素 3）确定选择的BTSA单独或与 联合治疗和研究新的生物标志物和凋亡调节机制。这项建议 将推进创新的治疗策略和治疗方法，并为最适合的环境提供信息， 靶向癌症中的BAX激活。

ABSTRACT Resistance to apoptosis is one of the hallmarks of cancer. Cancer cells circumvent cell death through the mitochondrial apoptosis pathway to ensure tumor growth, maintenance and resistance to current treatments. The mitochondrial apoptosis pathway is governed by the expression levels and interactions of the BCL-2 family proteins, which comprise the pro-apoptotic effector proteins BAX and BAK, the anti-apoptotic proteins e.g. BCL- 2, BCL-XL, MCL-1 and the pro-apoptotic BH3-only proteins. Cancer cells most commonly block mitochondrial apoptosis by upregulating the anti-apoptotic BCL-2 proteins that neutralize BH3-only proteins and activated BAX and BAK. Therefore, efforts have focused on the development of selective inhibitors of anti-apoptotic BCL-2 proteins to re-activate apoptosis. Inhibitors such as the FDA-approved Venetoclax rely on the function and availability of BH3-only proteins to activate BAX and BAK. However, in many tumors, BH3-only proteins can be downregulated, suppressed or deleted, making these tumors insensitive to these inhibitors and limiting their broader clinical application. We hypothesized that small-molecule direct activation of pro-apoptotic BAX via the BAX trigger site is an alternative and possibly complementary pharmacological strategy to promote apoptosis in cancer cells. This approach can promote BAX activation independently of BH3-only proteins and therefore should have the potential to overcome apoptosis blockade in resistant tumors. Our laboratory recently used unique structural and molecular insights and medicinal chemistry to develop a potent and selective compound, termed BAX Trigger Site Activator 1(BTSA1) that promotes BAX activation and induces mitochondrial dysfunction and apoptosis. Using BTSA1, we provided proof-of-concept for direct BAX activation as a therapeutic target in Acute Myeloid Leukemia and demonstrated that direct BAX activation is well tolerated in vivo. Here, we hypothesized to generate BTSA1 analogues with improved potency, oral bioavailability and pharmacokinetics. Our goal is to evaluate their activity and mechanism of action in diverse cancer models as single agents or combination treatments and investigate mechanisms of sensitivity and resistance to BAX activation and apoptosis. Moreover, we aim to identify a clinical candidate BAX activator with favorable cellular, pharmacological and safety properties. Therefore, we propose the following specific aims: 1) characterize potency, selectivity, pro-apoptotic activity, in vitro ADME/Tox and pharmacokinetic properties of BTSA1 analogues, 2) determine cellular efficacy and mechanism of action of 2nd generation BTSAs, including BTSA1.2, alone and in combination treatments using various leukemia and solid tumor cells and investigate determinants of sensitivity and resistance, 3) determine safety and therapeutic potential of select BTSAs alone or in combination therapy and investigate novel biomarkers and mechanisms of apoptosis regulation. This proposal will advance an innovative therapeutic strategy and therapeutics and inform the most suitable context for targeting BAX activation in cancer.