Small Molecule Inhibitors of BcI xL Survival Protein

描述：(申请人提供) 与Bcl2相关的生存蛋白使细胞产生广泛的耐药性 细胞凋亡诱导剂。在我们的实验室里，一部新奇的小作品 目前已经确定了Bclxl蛋白的分子配体，它可以抑制BclXL蛋白的 Bclxl的分子孔功能和选择性杀伤Bclxl，在较高浓度 剂量，表达BclNeg2的细胞数。我们初步观察到 表达bc1-xl的肝细胞系比同基因肝细胞系更敏感 控制细胞对已知的线粒体电子抑制物抗霉素A(AA)的作用 运输。一种对线粒体无作用的2-甲氧基抗菌素A类似物 呼吸对BclXL plus细胞仍表现出选择性毒性 线粒体。计算分子对接分析预测 抗霉素A符合分子上保守的疏水沟槽 Bc l-xl的表面。我们通过展示竞争性来确认这种互动 AA及其2-甲氧基衍生物与已知疏水沟槽的结合 BH3结构域多肽与重组Bc l-xl和Bc l-2蛋白的配基 来自支持细胞凋亡的二聚化伙伴Bak。最后，我们发现AA 抑制合成脂质体中Bclxl的成孔活性， 证明这种小配体可以直接抑制细胞的功能。 BCL-2相关的生存蛋白。这个应用程序的两个目标是调查 氨基酸与Bcl-XL疏水口袋结合的结构决定因素和 孔道抑制机理。 人造血细胞株的初步筛选及其细胞毒作用 抗霉素A提示骨髓瘤细胞株，包括多药耐药 亚系对AA和2-甲氧基AA敏感。几项已发表的研究已经 研究表明，骨髓瘤细胞的存活主要依赖于Bcl-xL，尽管 几种相关的抗细胞凋亡蛋白的表达。我们建议，使用 临床前模型，以测试多发性骨髓瘤是否特别 对Bcl-XL靶向治疗的易感性，并验证相关的 2-甲氧基AA在骨髓瘤细胞中的靶点， 将在三个具体施舍中解决的问题如下： 具体目标： 1.2-甲氧基抗霉素A3对小鼠的药效及毒理学评价 骨髓瘤和肝癌肿瘤模型。 2.抗霉素A抑制bc-1、xl的生化机制 成孔功能，包括分析Bcl-xl疏水基因突变 沟槽结合部位；抗霉素Bclxl络合物的X射线结晶学； 以及膜拓扑学研究。 3.确定内源性促凋亡二聚体对Eel-IF的作用 在抗霉素A的细胞毒机制中。

DESCRIPTION: (Provided by Applicant) The Bcl-2-related survival proteins confer cellular resistance to a wide range of apoptosis-inducing agents. In work can-led out in our labs, a novel small molecular ligand to the Bcl-xL protein has been identified, which inhibits the molecular pore function of Bcl-xL and selectively kills Bcl-xL and, at higher doses, Bcl neg2-expressing cells. We made the initial observation that Bcl-xL-expressing hepatocyte cell lines are more sensitive than isogenic control cells to antimycin A (AA), a known inhibitor of mitochondrial electron transport. A 2-methoxy antimycin A analog lacking effects on mitochondrial respiration still exhibited selective toxicity for Bcl-xL plus cells and mitochondria. Computational molecular docking analysis predicted that antimycin A conforms to a conserved hydrophobic groove on the molecular surface of Bcl-xL. We confirmed this interaction by showing competitive binding of AA and its 2-methoxy derivative with a known hydrophobic groove ligand to recombinant Bcl-xL and Bcl-2 proteins, a BH3 domain peptide derived from the pro-apoptotic dimerization partner, Bak. Finally, we found that AA inhibits the pore-forming activity of Bcl-xL in synthetic Liposomes, demonstrating that this small ligand can directly inhibit the function of Bcl-2-related survival proteins. Two aims of this application investigate the structural determinants of AA binding to the Bcl-xL hydrophobic pocket and mechanism of pore inhibition. Initial screening of human hematopoietic cell lines for cytotoxic effects of antimycin A indicates myeloma cell lines, including multi-drug resistant sublines, are sensitive to AA and 2-methoxy AA. Several published studies have shown that myeloma cell survival is predominantly dependent on Bcl-xL, despite the expression of several related anti-apoptotic proteins. We propose, using pre-clinical models, to test whether multiple myeloma is particularly susceptible to Bcl-xL -targeted therapies, and validate Bcl-xL as the relevant target of 2-methoxy AA in myeloma cells, The questions to be addressed In three specific alms are as follows: Specific Aims: 1. Evaluate efficacy and toxicology of 2-methoxy antimycin A3 in mouse myeloma and hepatoma tumor models. 2. Characterize biochemical mechanism of antimycin A inhibition of Bcl-,xL pore-forming function, including analysis of mutations in Bcl-xL hydrophobic groove binding site; x-ray crystallography of antimycin complex with Bcl-xL; and membrane topology studies. 3. Determine the role of endogenous pro-apoptotic dimer partners of Eel-if in the cytotoxic mechanism of antimycin A.