INHIBITORS OF CYCLIN DEPENDENT KINASES

生长调节途径的关键组分的功能丧失导致非程序性增殖，这是人类癌症的主要特征。该提案的总体目标是鉴定和表征可能用作开发癌症治疗剂的模型的有前景的先导化合物。这种抑制剂的潜在靶点包括那些提供生长所需功能的调节分子;其中一类蛋白质是细胞周期蛋白家族及其相关的催化亚基，细胞周期蛋白依赖性激酶（cdk）。细胞周期蛋白/cdk功能负责调节关键检查点，其允许细胞周期的不同阶段之间的穿越和转换，并且cdk 4和cdk 2功能的丧失已被证明在许多细胞模型中导致生长丧失和转化表型的逆转。该建议旨在鉴定肽序列和其他结构基序，其指定与细胞周期蛋白cdk活性的结合和抑制，以开发临床上有用的治疗剂。这将通过完成四个具体目标来实现。第一个目标是从噬菌体展示肽库中筛选出与cyclin/cdk亚基相互作用的肽段，并利用其序列从噬菌体展示肽库中开发靶向组合库。在第二个目标中，与细胞周期蛋白/cdk亚基特异性相互作用的肽，基于其保守结构基序的第二代组合文库和在项目1中开发的另外的组合文库将通过高通量生物化学测定来筛选，以鉴定那些能够抑制酶活性的分子。第三个目标是利用人肿瘤细胞培养模型来确定全细胞中潜在抑制剂的特异性和选择性。在第四个目标中，将在细胞培养物和裸鼠中的肿瘤发生模型中对已证明在全细胞中有效的那些化合物进行分析，以评估它们抑制生长和逆转转化的能力。

The loss of function of key components of growth regulatory pathways results in unprogrammed proliferation, the primary characteristic of human cancer. The overall goal of this proposal is to identify and characterize promising lead compounds that may be utilized as models to develop therapeutic agents for cancer. Potential targets for such inhibitors include those regulatory molecules that provide functions necessary for growth; one such class of proteins is the cyclin family and their associated catalytic subunits, the cyclin-dependent kinases (cdk). Cyclin/cdk functions are responsible for regulating key checkpoints that allow traverse through and transitions between different stages of the cell cycle, and loss of cdk4 and cdk2 functions have been demonstrated to result in a loss of growth and reversion of a transformed phenotype in a number of cell models. This proposal seeks to identify peptide sequences and other structural motifs that specify binding to and inhibition of cyclin-cdk activity to develop clinically useful therapeutics. This will be accomplished through the completion of four Specific Aims. In the first aim, peptides that interact with cyclin/cdk subunits will be identified from a phage of display peptide library, and their sequences will be utilized to develop targeted combinatorial from a phage display peptide library, and their sequences will be utilized to develop targeted combinatorial libraries. In the second aim, peptides that interact specifically with cyclin/cdk subunits, second generation combinatorial libraries based on their conserved structural motifs and additional combinatorial libraries developed in Project 1 will be screened by a high throughput biochemical assay to identify those molecules capable of inhibiting enzymatic activity. The third aim will utilize human tumor cell culture models to ascertain the specificity and selectivity of potential inhibitors in whole cells. In the fourth aim, those compounds which have proven to be effective in whole cells will be subjected to analysis in tumorigenic models, both in cell cultures and in nude mice, to assess their ability to inhibit growth and revert transformation.