Modulation of BAK in Lung Cancer Therapeutics

大多数肺癌患者由于基因改变和对传统疗法的抵抗而预后不良 治疗。开发新的、更有效的药物对于改善患者的预后至关重要 非小细胞肺癌（NSCLC）和小肺癌（SCLC）。 BAK 是一个关键的多域 Bcl2 家族中的促凋亡分子，是细胞凋亡所需的。 因为比克通常是 一种完整的线粒体外膜蛋白，而 BAX 需要在 作为一种细胞凋亡刺激，BAK 激活可能更容易处理。 BAK同二聚体或异二聚体的形成是 诱导细胞凋亡的重要机制。 Bcl-XL 和 Mcl-1 可以与 BAK 结合，这可能是 处于“引发”构象，其 BH3 结构域暴露，而在凋亡诱导的细胞中，只有 BH3 的蛋白质 取代抗凋亡异二聚体中的 BAK。然后游离的 BAK 形成寡聚物，引发 线粒体外膜的透化和细胞色素 c 的释放导致细胞凋亡。 BH3结合口袋位于BAK α2和β3之间，这是设计或筛选的理想位点 BAK 激活剂用于开发潜在的新型抗癌药物。我们选择BH3域结合 BAK 的口袋 (aa75-90) 作为使用 UCSF DOCK 6.1 筛选小分子的对接位点 程序套件和 NCI 化学库数据库。我们发现了两种新型 BAK 激活剂（BKA-073 和 BKA-758）与正常人支气管细胞相比，对肺癌细胞表现出选择性凋亡作用 上皮细胞。荧光偏振测定表明 BKA-073 和 BKA-758 优先与 BAK 结合 体外抑制常数 (Ki) 值为纳摩尔水平的蛋白质，但不与其他 Bcl2 家族结合 成员（即 BAX 或 Bcl2）。先导化合物 BKA-758 对肺癌具有有效的抗肿瘤活性 来自肺癌细胞系或患者来源的异种移植模型的异种移植物。我们假设 BKA 可以通过特异性靶向 BAK 的 BH3 来特异性激活肿瘤细胞中 BAK 的促凋亡功能 结合口袋，从而抑制肺癌。为了检验这个假设，我们确定了两个 具体目标： (1) 确定 BAK 激动剂 (BKA) 激活促凋亡细胞的机制 BAK的功能并诱导人肺癌细胞凋亡； (2) 判断BKA是否化合物 体内抑制小肺癌（SCLC）和非小细胞肺癌（NSCLC）的生长。研究将 测试 BKA 在患者来源的异种移植物、抗辐射肺癌和基因工程中的效力 肺癌动物模型。确定“动态 BH3 分析”是否可以预测以下药物的抗肿瘤功效 BKA 体内化合物。根据我们提出的研究，预计新型一流的 BAK 激动剂 作为一类全新的抗肺癌药物，将被开发用于肺癌治疗。

Most patients with lung cancer have a poor prognosis due to genetic alterations and resistance to conventional therapy. The development of novel and more effective drugs is critical to improve the prognosis of patients with both non-small cell lung cancer (NSCLC) and small lung cancer (SCLC). BAK is a key multidomain proapoptotic molecule in the Bcl2 family, which is required for apoptotic cell death. Because BAK is normally an integral outer mitochondrial membrane protein, whereas BAX requires translocation from the cytosol after an apoptotic stimulus, BAK activation may be more tractable. The formation of BAK homo- or heterodimers is an important mechanism in the induction of apoptosis. Bcl-XL and Mcl-1 can bind to BAK, which is presumably in a ‘primed’ conformation with its BH3 domain exposed, while in apoptosis-induced cells, a BH3-only protein displaces BAK from the anti-apoptotic heterodimer. The free BAK then forms an oligomer that elicits the permeabilization of the mitochondrial outer membrane and the release of cytochrome c leading to apoptosis. The BH3 binding pocket is located between BAK α2 and β3, which is an ideal site for the design or screening of BAK activators for the development of potential new anti-cancer agents. We chose the BH3 domain binding pocket (aa75-90) of BAK as a docking site for the screening of small molecules using the UCSF DOCK 6.1 program suite and the NCI chemical library database. We discovered two novel BAK activators (BKA-073 and BKA-758) that exhibit selective apoptotic effect in lung cancer cells as compared to normal human bronchial epithelial cells. Fluorescence polarization assay reveals that BKA-073 and BKA-758 preferentially bind to BAK protein with inhibitory constant (Ki) values at nanomolar levels in vitro but do not bind to other Bcl2 family members (i.e. BAX or Bcl2). The lead compound BKA-758 has potent antitumor activity against lung cancer in xenografts derived from lung cancer cell lines or patient-derived xenograft models. We hypothesize that BKA(s) may specifically activate the proapoptotic function of BAK in tumor cells by specifically targeting its BH3 binding pocket, which leads to suppression of lung cancer. To test this hypothesis, we have identified two specific aims: (1) To determine the mechanism(s) by which BAK agonists (BKAs) activate the proapoptotic function of BAK and induce apoptosis in human lung cancer cells; (2) To determine whether BKA compounds suppress the growth of small lung cancer (SCLC) and non-small cell lung cancer (NSCLC) in vivo. Studies will test the potency of BKAs in patient-derived xenografts, radioresistant lung cancer and genetically engineered lung cancer animal models. Determine whether the “dynamic BH3 profiling” predicts the antitumor efficacy of BKA compounds in vivo. Based on our proposed studies, it is expected that novel first-in-class BAK agonists as an entirely new class of anti-lung cancer drugs will be developed for lung cancer therapy.