High Resol.3-D Structures of Sm. Molecule Inhibitors with Bcl-2 and Bcl-xL by X-r

抗凋亡Bcl-2家族成员Bcl-2和Bcl-xL在许多癌细胞中过表达，最终导致细胞永生和对触发凋亡途径的常规癌症疗法的抗性。因此，我们相信开发特异性靶向这些抗凋亡蛋白的小分子化合物将降低细胞对癌症疗法的抗性。目的是开发和合成新型的、高效的和选择性的Bcl-xL和Bcl-2小分子抑制剂，并在体外和体内测试它们在癌细胞中的治疗潜力。该提案的一部分将通过提供对抑制剂结合的原子详细分析来补充其他提案，所述抑制剂结合需要增加小分子化合物对Bcl-2和BclxL的效力和选择性。对于我们的结构研究，我们将采用核磁共振和X射线晶体学的补充技术。为了实现我们的目标，我们提出了以下四个具体目标：目标1：使用NMR筛选方法确认抑制剂与Bcl-xL和Bcl-2的BH 3结合位点的结合;目标2：使用多维核磁共振技术确定与抑制剂复合的Bcl-2和Bcl-xL的三维结构。 NMR方法;目标3：测定高效小分子化合物的高分辨率X射线晶体结构 与Bcl-xL复合的分子抑制剂;目的4.：通过定点诱变和生化结合研究分析Bcl-2和Bcl-xL残基对抑制剂结合至关重要。人类BclxL和Bcl-2与来自每类化合物的代表复合的结构研究将有助于鉴定对抑制剂结合至关重要的残基，并为设计每类内的新抑制剂提供基础。BH 3结合沟中的非保守残基以及参与结合位点结构稳定性的残基的突变分析可以提供对蛋白质对抑制剂的特异性的洞察。这些研究结合与抑制剂复合的Bcl-xL和Bcl-2的结构测定，将有助于鉴定抑制剂结合的关键残基，并为设计新的抑制剂提供基础， 对这些个体目标的特异性更强。

The anti-apoptotic Bcl-2 family members, Bcl-2 and Bcl-xL, are over-expressed in many cancer cells, ultimately leading to cell immortality and resistance to the conventional cancer therapies that trigger the apoptotic pathway. Therefore, we believe developing small molecule compounds that specifically target these anti-apoptotic proteins will reduce the cellular resistance to cancer therapies. The objectives are to develop and synthesize novel, highly potent and selective small molecule inhibitors to Bcl-xL and Bcl-2 and test them in vitro and in vivo for their therapeutic potential in cancer cells. Part of this proposal will complement the others by providing the atomic detailed analysis of inhibitor binding needed to increase potency and selectivity of the small molecule compounds toward Bcl-2 and BclxL. For our structural studies, we will employ the complimentary techniques of NMR and X-ray crystallography. To accomplish our goal, we propose to do the following four Specific Aims: Aim 1: Confirmation of inhibitor binding to the BH3 binding site of Bcl-xL and Bcl-2 using NMR screening methods; Aim 2: Determination of three-dimensional structures of Bcl-2 and Bcl-xL complexed with inhibitors by multidimensional NMR methods; Aim 3: Determination of high-resolution X-ray crystal structures of potent small molecule inhibitors in complex with Bcl-xL; Aim 4.: Analysis of Bcl-2 and Bcl-xL residues crucial for inhibitor binding through site-directed mutagenesis and biochemical binding studies. Structural studies of human BclxL and Bcl-2 in complex with representatives from each class of compounds will aid in identifying residues crucial for inhibitor binding and provide the foundation for designing new inhibitors within each class. Mutational analysis of non-conserved residues in the BH3 binding groove as well as residues involved in the structural stability of the binding site may offer insight into the protein's specificity for inhibitors. These studies in conjunction with the structural determination of Bcl-xL and Bcl-2, in complex with inhibitors, will aid in identifying key residues for inhibitor binding and provide the foundation for designing new inhibitors having greater specificity toward these individual targets.