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

抗凋亡的Bcl2家族成员Bcl2和Bclxl在许多肿瘤细胞中过度表达，最终导致细胞永生，并对触发细胞凋亡途径的传统癌症治疗产生抵抗。因此，我们相信，开发专门针对这些抗凋亡蛋白的小分子化合物将降低细胞对癌症治疗的抵抗力。本研究的目的是开发和合成新型、高效和选择性的抗BclxL和Bcl2的小分子抑制剂，并在体内外测试它们对癌细胞的治疗潜力。这项建议的一部分将通过提供所需的抑制剂结合的原子详细分析来补充其他建议，以提高小分子化合物对Bcl-2和BclxL的效力和选择性。在我们的结构研究中，我们将使用核磁共振和X射线结晶学的互补技术。为了实现我们的目标，我们提出了以下四个具体的目标：目标1：用核磁共振筛选方法确定与BH3结合的BH3结合位点的抑制剂；目标2：用多维方法确定与抑制剂络合的BH3的三维结构 核磁共振方法；目标3：有效小分子的高分辨X射线晶体结构的测定 目的4.通过定点突变和生化结合研究，分析对抑制剂结合至关重要的Bcl2和Bclxl残基。对每类化合物代表的人BclxL和Bcl2复合体的结构研究将有助于识别对抑制剂结合至关重要的残基，并为在每类化合物中设计新的抑制剂提供基础。对BH3结合槽中非保守残基以及与结合位点结构稳定性有关的残基进行突变分析，可能有助于深入了解该蛋白质对抑制剂的特异性。这些研究结合抑制剂复合体中Bclxl和Bcl2的结构测定，将有助于确定与抑制剂结合的关键残基，并为设计具有以下特性的新型抑制剂提供基础 对这些单独的目标有更大的特异性。

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.