Modulation of BAK in Lung Cancer Therapeutics

大多数肺癌患者由于遗传变异和对常规化疗的抵抗而预后不良。 疗法新的和更有效的药物的开发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）。 巴克是一个关键的多域 Bcl 2家族中的促凋亡分子，其是凋亡性细胞死亡所需的。 因为巴克通常 一个完整的线粒体外膜蛋白，而BAX需要易位后，从胞质溶胶 作为一种凋亡刺激，巴克激活可能更容易处理。 巴克同二聚体或异二聚体的形成是 是诱导细胞凋亡的重要机制。Bcl-XL和Mcl-1可以与BAK结合，推测BAK是 在暴露其BH 3结构域的“引发”构象中，而在凋亡诱导的细胞中， 从抗凋亡异二聚体中置换巴克。然后游离巴克形成低聚物， 线粒体外膜的透化和细胞色素c的释放导致细胞凋亡。 BH 3结合口袋位于巴克α2和β3之间，这是设计或筛选的理想位点 巴克激活剂的研究，用于开发潜在的新抗癌药物。我们选择了BH 3结构域结合 巴克的口袋（aa 75 -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化学图书馆数据库。我们发现了两种新的巴克激活剂（BKA-073和 BKA-758），与正常人支气管肺癌细胞相比，其在肺癌细胞中表现出选择性凋亡作用 上皮细胞荧光偏振分析显示BKA-073和BKA-758优先与巴克结合 在体外具有纳摩尔水平的抑制常数（Ki）值但不与其他Bcl 2家族结合的蛋白质 成员（即BAX或Bcl 2）。先导化合物BKA-758对肺癌具有有效的抗肿瘤活性， 来源于肺癌细胞系的异种移植物或来源于患者的异种移植物模型。我们假设 BKA（s）可通过特异性靶向其BH 3而特异性激活肿瘤细胞中巴克的促凋亡功能 结合口袋，这导致抑制肺癌。为了验证这一假设，我们确定了两个 具体目的：（1）确定巴克激动剂（BKAs）激活促凋亡细胞的机制， 巴克的功能和诱导人肺癌细胞凋亡;（2）确定BKA化合物是否 在体内抑制小肺癌（SCLC）和非小细胞肺癌（NSCLC）的生长。研究将 测试BKAs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BH 3谱”是否预测了 体内BKA化合物。基于我们提出的研究，预期新型一流的巴克激动剂 作为一种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.