Development of Small Molecule Antagonists of Bcl2/BclXL

细胞凋亡的逃逸是肿瘤进展和耐药的主要机制。反- 凋亡的Bcl2家族蛋白，Bcl2和Bclxl，是最有效的细胞凋亡抑制因子，是 在各种类型的人类癌症中频繁上调。Bcl2/Bclxl的抗细胞凋亡作用依赖于 至少在一定程度上，取决于它们与促凋亡的Bcl-2家族成员Bax的异源二聚体和抑制能力 还有贝克。诱变研究表明，BH3保守的结构域在促凋亡的Bcl-2中 家族蛋白对于它们的细胞死亡诱导功能以及它们与Bcl2和Bclxl的结合都是至关重要的。 对Bc l-xl/BH3复合物的结构分析表明，a-螺旋BH3多肽与 由BH1-3结构域形成的疏水裂隙。此外，还表明，合成的 包含各种促凋亡的Bcl-2家族成员BH3结构域的多肽足以 诱导癌细胞凋亡。这些研究表明，小分子BH3-BH3- 模拟药物是开发新型抗癌药物的一种很有前途的策略。为此，我们 在此基础上，合理设计合成了一系列低分子量的Bcl-xL拮抗剂 三苯基和对苯二甲酰胺支架，以模拟Bak-BH3a-螺旋。初步筛查发现了几个 在亚微摩尔区或高纳米摩尔区具有结合亲和力的先导化合物，通过荧光 用荧光标记的Bak-BH3多肽和纯化的Bclxl蛋白进行体外偏振(FP)分析。 计算对接模拟和核磁共振分析表明，这些合成的缓蚀剂针对的是-BH3- Bc l-xl表面出现结合裂隙。此外，活性化合物还能干扰Bclxl的相互作用。 在体外和全细胞内与Bax结合，通过细胞凋亡和裸鼠肿瘤消退而导致细胞死亡。 该项目的目标是开发小分子，通过特定的途径诱导癌细胞凋亡。 破坏BHS介导的促凋亡蛋白和抗凋亡蛋白之间的异二聚化。 具体目标是：1)使用合理的设计以及高通量筛选(NTS)来鉴定 BH3a-螺旋模拟物；2)确定在特定条件下鉴定的化合物的效力和专一性 目的1阻断BHS介导的BHS介导的Bcl2促凋亡和抗凋亡成员间的异二聚化 在无细胞和完整细胞中都有一个家族；3)测定Bc l/BH3的活性和选择性 在特定目标2中发现的干扰物可诱导细胞凋亡并抑制培养细胞中的肿瘤生长 在小鼠模型中也是如此，并验证其潜在的生化机制。

The escape of apoptosis is a major mechanism for cancer progression and drug resistance. The anti- apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, are among the most effective inhibitors of apoptosis and are frequently upregulated in various types of human cancer. The anti-apoptotic function of Bcl-2/Bcl-xL depends, at least in part, on their ability to heterodimerize with and inhibits the pro-apoptotic Bcl-2 family members Bax and Bak. Mutagenesis studies have shown that the conserved BH3 domain within the pro-apoptotic Bcl-2 family proteins is critical for their cell death-inducing function and also for their binding to Bcl-2 and Bcl-xL. Structural analyses of the Bcl-xL/BH3 complexes revealed that the a-helical BH3 peptides bind to the hydrophobic cleft formed by the BH1-3 domains of Bcl-xL. Moreover, it has been shown that synthesized peptides encompassing the BH3 domains of various pro-apoptotic Bcl-2 family members are sufficient to induce apoptosis of cancer cells. These studies suggest that targeting of Bcl-2/Bcl-xL by small molecule BH3- mimitics is a promising strategy for the development of a new class of anticancer drugs. To this end, we have rationally designed and synthesized a series of low-molecular-weight Bcl-xL antagonists based on the terphenyl and terephthalamide scaffolds to mimic the Bak-BH3 a-helix. Initial screens have identified several lead compounds, with binding affinities in the sub micromolar or high nanomolar region, by fluorescence polarization (FP) assay using fluoresceih-labeled Bak-BH3 peptide and purified Bcl-xL protein in vitro. Computational docking simulation and NMR analyses revealed that these synthetic inhibitors target the-BH3- binding cleft on the surface of Bcl-xL. Moreover, the active compounds can disrupt the interaction of Bcl-xL with Bax in vitro and in whole cells and cause cell death by apoptosis and tumor regression in nude mice. The goal of this project is to develop small molecules that induce apoptosis of cancer cells specifically by disrupting the BHS-mediated heterodimerization between pro- and anti-apoptotic proteins of the Bcl-2 family. The Specific Aims are: 1) To use rational design as well as high throughput screening (NTS) to identify BH3 a-helical mimics; 2) To determine the potency and specificity of the compounds identified under Specific Aim 1 to disrupt the BHS-mediated heterodimerization between pro- and anti-apoptotic members of the Bcl-2 family both in cell-free and in intact cells; 3) To determine the potency and selectivity of the Bcl/BH3 disrupters identified in Specific Aim 2 to induce apoptosis and to suppress tumor growth in cultured cells as well as in mouse models and verify the underlying biochemical mechanisms.