Mechanism of Action of Folate Antagonist

描述（由申请人提供）：本修订版更新申请的目的是详细了解两种关键酶（二氢叶酸还原酶（DHFR）和胸苷酸合成酶（TS））的转录和翻译调控，这两种酶分别是已确立的抗癌药物甲氨蝶呤和5-氟尿嘧啶的靶点。驱动这些研究的假设是，这种理解将为药物开发和更合理地使用这些药物（单独或组合）产生新的靶点。有三个具体目标： 特定的Aim 1描述了我们对DHFR翻译调控的研究。生物物理测量（荧光测定法、圆二色性和等温量热法）和X射线晶体学将用于研究DHFR与其同源mRNA的结合，以及与蛋白质结合所涉及的mRNA区域。将评价DHFR蛋白的单个氨基酸和区域的突变分析，以揭示DHFR蛋白参与其与DHFR mRNA结合的区域。该信息将用于产生在MTX处理后阻断与DHFR结合的肽适体。 特异性目标2将进一步表征我们已经显示的抑制E2 F与其在DNA中的识别序列结合并具有细胞毒性活性的肽。与罗格斯大学的Breslauer博士及其同事合作，计划优化肽序列以提高稳定性和增加结合力。将使用对这些转录因子中的每一种特异性的siRNA来研究下调活化E2 F（E2 F 1、2、3）的效果。该特定目的的一个目标是测量这些转录因子中的每一种单独和组合下调对DHFR、TS和核糖核苷酸还原酶水平的影响，以及对这些酶的特异性抑制剂的相应敏感性。 具体目标3进一步探讨了我们以前的观察，即在具有非功能性p53的细胞中p14 ARF的强制表达下调DHFR。使用p53、p19 ARF和两者均无效的MEF细胞将用于在更明确的系统中测试这些蛋白质对DHFR水平的功能。

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.