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

抗凋亡的Bcl-2家族成员Bcl-2和Bcl-xL在许多癌细胞中过度表达，最终导致细胞不朽并对触发凋亡途径的常规癌症治疗产生耐药性。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耐药性。目标是开发和合成新的、高效的、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对癌细胞的治疗潜力。该提案的一部分将通过提供抑制剂结合所需的原子详细分析来补充其他内容，以提高小分子化合物对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射线晶体学的互补技术。为了实现我们的目标，我们提出以下四个具体目标：目标1：利用核磁共振筛选方法确认抑制剂与Bcl-xL和Bcl-2的BH3结合位点的结合；目的2：用多维度法测定Bcl-2和Bcl-xL与抑制剂络合的三维结构

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.