dUTPase as a Target for Drug Discovery

描述（由申请人提供）：本提案的总体目标是进一步验证dUTR作为药物发现的靶点。dUTP催化dUTP水解形成dUMP和PPi。该反应有效地从DNA生物合成途径中去除dUTP，从而防止有害的尿嘧啶错误掺入DNA。广泛使用的胸苷酸代谢抑制剂（即5-FU和甲氨蝶呤）可诱导TTP池严重耗竭，在某些情况下，还可诱导dUTP池同时蓄积，导致尿嘧啶反复错误掺入DNA，从而导致DNA损伤和细胞死亡。尽管一些癌细胞系能够在TS抑制后积累dUTP池，但其他癌细胞系不能;导致肿瘤中升高的dUTP活性阻止dUTP积累导致耐药性的假设。实际上，肿瘤标本中dUTR的过表达与转移性结肠癌中对TS导向化疗的无应答相关（拉德纳，et al. 2000）。dUTP酶是dUTP库的关键调节因子，代表了经证实的临床实用性途径中有希望的替代治疗靶点。抑制dUTR与传统药物的组合可以提供一种新的方法，以充分利用胸苷酸生物合成作为化疗靶点。我们假设，有效的dUTR抑制剂将增强传统TS抑制剂的疗效，并克服肿瘤内dUTR过度表达导致的耐药性。该提案的主要目的是下调一系列癌细胞系中的dUTR表达，并验证抑制活性对靶向胸苷酸生物合成的药剂的功效的影响。具体目标1：降低细胞dUTR活性是否使人癌细胞对广泛使用的胸苷酸和叶酸代谢抑制剂敏感？具体目标1研究了dUTR下调对抑制胸苷酸合成酶和叶酸代谢的化疗药物毒性的影响。我们的方法利用siRNA介导的dUTR活性下调，初步数据表明我们有能力利用这种技术。具体目标二：TS抑制剂和dUTR活性降低之间的协同作用是否与尿嘧啶错误掺入的生化终点相关？机制分析将用于评价尿嘧啶错误掺入途径的生化终点。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to further validate dUTPase as a target for drug discovery. dUTPase catalyzes the hydrolysis of dUTP to form dUMP and PPi. This reaction effectively removes dUTP from the DNA biosynthetic pathway, thereby preventing detrimental uracil misincorporation into DNA. Broadly utilized inhibitors of thymidylate metabolism (i.e. 5-FU and methotrexate) induce a severe depletion of TTP pools and in some cases, a concurrent accumulation of dUTP pools that results in the iterative misincorporation of uracil into DNA, leading to DNA damage and cell death. Although some cancer cell lines are able to accumulate dUTP pools upon TS inhibition, others do not; leading to the hypothesis that elevated dUTPase activity in tumors prevents dUTP accumulation resulting in drug resistance. Indeed, overexpression of dUTPase in tumor specimens is associated with non-response to TS-directed chemotherapy in metastatic colon cancer (Ladner, et al. 2000). dUTPase, the key regulator of dUTP pools, represents a promising alternative therapeutic target within a pathway of proven clinical utility. Inhibition of dUTPase in combination with traditional agents may provide a novel approach to fully exploit thymidylate biosynthesis as a chemotherapeutic target. We hypothesize that an effective dUTPase inhibitor will enhance the efficacy of traditional TS inhibitors and overcome drug resistance that results from overexpression of intratumoral dUTPase. The main objective of this proposal is to down regulate dUTPase expression in a series of cancer cell lines and validate the impact of inhibited activity on the efficacy of agents that target thymidylate biosynthesis. Specific Aim 1: Does lowering cellular dUTPase activity sensitize human cancer cells to widely utilized inhibitors of thymidyate and folate metabolism? Specific Aim 1 investigates the effect of dUTPase down regulation on the toxicity of chemotherapeutic agents that inhibit thymidylate synthase and folate metabolism. Our approach utilizes siRNA-mediated down regulation of dUTPase activity, and preliminary data demonstrating our ability to utilize this technique is presented. Specific Aim 2: Does the synergy between TS inhibitors and lowered dUTPase activity correlate with the biochemical endpoints of uracil misincorporation? A mechanistic analysis will be used to evaluate the biochemical enpoints underlying the uracil misincorporation pathway.