Mechanism of Action of Folate Antagonist

此修订续订申请的目标是详细了解转录和翻译 二氢叶酸还原酶(DHFR)和胸苷酸合成酶(TS)这两种关键酶的调节 作为公认的抗癌药物的靶标，分别是甲氨蝶呤和5-氟尿嘧啶。假说 推动这些研究的是，这种理解将为药物开发产生新的目标和更多 合理使用这些制剂，单独使用或联合使用。具体目标有三个： Speeifie Aim#1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射线结晶学将用于研究 DHFR与其同源信使核糖核酸的结合，以及与蛋白质结合的信使核糖核酸区域。 将对单一氨基酸和DHFR蛋白区域的突变分析进行评估，以揭示 DHFR蛋白参与其与DHFR基因的结合。这些信息将被用于生成多肽 在MTX治疗后阻断与DHFR结合的适配子。 特殊目标#2将进一步表征我们已经显示的抑制E2F与其识别结合的多肽 DNA序列，并具有细胞毒活性。优化多肽序列以提高稳定性和 增加绑定是与罗格斯大学的布雷斯劳尔博士和同事合作计划的。这个 将使用针对每个E2F(E2F 1，2，3)的siRNA来研究下调激活E2F(E2F 1，2，3)的效果 这些转录因子。这一特定目标的一个目标是衡量每一项下调的影响 在这些转录因子中，单独和联合作用于DHFR、TS和核苷酸还原酶水平， 以及对这些酶的特定抑制剂的相应敏感性。 具体目标#3进一步探索了我们之前的观察结果，即在细胞中强制表达p14ARF 与无功能的P53一起下调DHFR。使用p53、p19 ARF和两者均为空的MEF细胞 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。

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: Speeifie aim#1 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.