Mechanism of Action of Folate Antagonist

描述(申请人提供)：本次修订的续签申请的目标是详细了解二氢叶酸还原酶(DHFR)和胸苷酸合酶(TS)这两种关键酶的转录和翻译调节，这两种酶分别是公认的抗癌药物甲氨蝶呤和5-氟尿嘧啶的靶标。推动这些研究的假设是，这种理解将为药物开发和这些药物的更合理使用产生新的靶点，无论是单独使用还是联合使用。具体目标有三个： 特异性Aim1描述了我们对DHFR翻译调控的研究。生物物理测量(荧光法、圆二色谱和等温量热法)和X射线结晶学将被用来研究DHFR与其同源mRNA的结合，以及与蛋白质结合的mRNA区域。对单个氨基酸和DHFR蛋白区域的突变分析将被评估，以揭示DHFR蛋白与DHFR mRNA结合所涉及的区域。这些信息将被用来产生多肽适体，在MTX治疗后阻断与DHFR的结合。 特定目的2将进一步表征我们已经证明抑制E2F与其DNA中的识别序列结合并具有细胞毒活性的多肽。与罗格斯大学的布雷斯劳尔博士和同事们合作，计划对多肽序列进行优化，以提高稳定性和增加结合。将使用针对每个转录因子的siRNA来研究下调激活E2F(E2F 1，2，3)的效果。这一特定目的的一个目的是测量这些转录因子中的每一个单独和联合下调对DHFR、TS和核糖核苷酸还原酶水平的影响，以及对这些酶的特定抑制剂的相应敏感性。 特定目的3进一步探索了我们之前的观察结果，即在具有无功能的p53的细胞中强制表达p14ARF下调了DHFR。使用p53、p19ARF和两者均为空的MEF细胞将被用来在更明确的系统中测试这些蛋白质在dhfr水平上的功能。

DESCRIPTION (provided by applicant): The goal of this revised renewal application is to understand in detail the transcriptional and translational regulation of two key enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS), that are targets for well established anticancer agents, methotrexate and 5- fluorouracil respectively. The hypothesis that drives these studies is that this understanding will generate new targets for drug development and more rational use for these agents, alone or in combination. There are three specific aims: Specific Aim1 describes our studies of the translational regulation of DHFR. Biophysical measurements (fluorometry,circular dichroism and isothermal calorimetry) and x-ray crystallography will be used to study the binding of DHFR to its cognate mRNA, and regions of mRNA involved in binding to the protein. Mutational analysis of single amino acids and regions of DHFR protein will be evaluated to reveal areas of DHFR protein involved in its binding to DHFR mRNA. This information will be used to generate peptide aptamers that block the binding to DHFR following MTX treatment. Specific Aim 2 will further characterize a peptide that we have shown inhibits E2F binding to its recognition sequence in DNA and has cytoxic activity. Optimization of the peptide sequence to improve stability and increase binding are planned in collaboration with Dr. Breslauer and colleagues of Rutgers University. The effect of down regulation of activating E2Fs (E2F 1,2,3) will be studied using siRNAs specific to each of these transcription factors. A goal of this specific aim is to measure the effect of the down regulation of each of these transcription factors, alone and in combination on levels of DHFR, TS and ribonucleotide reductase, and corresponding sensitivity to specific inhibitors of these enzymes. Specific Aim 3 explores further our previous observations, namely that forced expression of p 14ARF in cells with non-functional p53 down regulates DHFR. The use of MEF cells null for p53, p19 ARF and both will be utilized to test in a more well defined system the function of these proteins on DHFR levels.