Department of Radiation Oncology, Emory University School of Medicine and Winship Cancer Institute of Emory University, Atlanta, Georgia 30322, USA.
Institute for Systems Biology, Seattle, WA 98109, USA.
Theranostics. 2021 Jul 25;11(17):8500-8516. doi: 10.7150/thno.60349. eCollection 2021.
Bak is a major proapoptotic Bcl2 family member and a required molecule for apoptotic cell death. High levels of endogenous Bak were observed in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Increased Bak expression was correlated with poor prognosis of NSCLC patients, suggesting that Bak protein is an attractive target for lung cancer therapy. The BH3 domain functions as death domain and is required for Bak to initiate apoptotic cell death. Thus, the BH3 domain is attractive target for discovery of Bak agonist. The BH3 death domain binding pocket (aa75-88) of Bak was chosen as a docking site for screening of small molecule Bak activators using the UCSF DOCK 6.1 program suite and the NCI chemical library (300,000 small molecules) database. The top 500 compounds determined to have the highest affinity for the BH3 domain were obtained from the NCI and tested for cytotoxicity for further screening. We identified a small molecule Bak activator BKA-073 as the lead compound. The binding affinity of BKA-073 with Bak protein was analyzed by isothermal titration calorimetry (ITC) assay. BKA-073-mediated Bak activation via oligomerization was analyzed by a cross-linking with Bis (maleimido) hexane (BMH). Sensitivity of BKA-073 to lung cancer cells in vitro was evaluated by dynamic BH3 profiling (DBP) and apoptotic cell death assay. The potency of BKA-073 alone or in combination with radiotherapy or Bcl2 inhibitor was evaluated in animal models. We found that BKA-073 binds Bak at BH3 domain with high affinity and selectivity. BKA-073/Bak binding promotes Bak oligomerization and mitochondrial priming that activates its proapoptotic function. BKA-073 potently suppresses tumor growth without significant normal tissue toxicity in small cell lung cancer (SCLC) and NSCLC xenografts, patient-derived xenografts, and genetically engineered mouse models of mutant KRAS-driven cancer. Bak accumulates in radioresistant lung cancer cells and BKA-073 reverses radioresistance. Combination of BKA-073 with Bcl-2 inhibitor venetoclax exhibits strong synergy against lung cancer in vivo. Development of small molecule Bak activator may provide a new class of anticancer agents to treat lung cancer.
Bak 是一种主要的促凋亡 Bcl2 家族成员，也是细胞凋亡所必需的分子。在小细胞肺癌 (SCLC) 和非小细胞肺癌 (NSCLC) 细胞系中均观察到内源性 Bak 水平升高。Bak 表达增加与 NSCLC 患者预后不良相关，表明 Bak 蛋白是肺癌治疗的一个有吸引力的靶点。BH3 结构域作为死亡结构域，是 Bak 启动细胞凋亡所必需的。因此，BH3 结构域是发现 Bak 激动剂的有吸引力的靶点。使用 UCSF DOCK 6.1 程序套件和 NCI 化学文库 (30 万个小分子) 数据库，选择 Bak 的 BH3 死亡结构域结合口袋 (aa75-88) 作为筛选小分子 Bak 激活剂的对接位点。从 NCI 获得了确定与 BH3 结构域结合亲和力最高的前 500 种化合物，并进行细胞毒性测试以进一步筛选。我们鉴定出一种小分子 Bak 激活剂 BKA-073 作为先导化合物。通过等温滴定量热法 (ITC) 分析测定 BKA-073 与 Bak 蛋白的结合亲和力。通过 Bis(maleimido)hexane (BMH) 交联分析 BKA-073 介导的 Bak 寡聚化激活。通过动态 BH3 谱分析 (DBP) 和凋亡细胞死亡测定评估 BKA-073 对体外肺癌细胞的敏感性。在动物模型中评估 BKA-073 单独或与放射治疗或 Bcl2 抑制剂联合的疗效。我们发现 BKA-073 以高亲和力和选择性与 Bak 的 BH3 结构域结合。BKA-073/Bak 结合促进 Bak 寡聚化和线粒体引发，从而激活其促凋亡功能。BKA-073 可有效抑制肿瘤生长，而对小细胞肺癌 (SCLC) 和 NSCLC 异种移植、患者来源的异种移植和突变型 KRAS 驱动的癌症的基因工程小鼠模型中的正常组织无明显毒性。Bak 在放射抗性肺癌细胞中积累，BKA-073 逆转放射抗性。BKA-073 与 Bcl2 抑制剂 venetoclax 联合具有很强的体内抗肺癌协同作用。开发小分子 Bak 激活剂可能为治疗肺癌提供一类新的抗癌药物。
