dUTPase as a Target for Drug Discovery

描述(由申请人提供)：这项提案的总体目标是进一步验证dUTPase作为药物发现目标的有效性。DUTP酶催化dUTP的水解形成DUMP和PPI。该反应有效地将dUTP从DNA生物合成途径中移除，从而防止有害的尿嘧啶错误结合到DNA中。广泛使用的胸苷代谢抑制剂(即5-FU和甲氨蝶呤)会导致TTP池的严重枯竭，在某些情况下，dUTP池的同时积累会导致尿嘧啶反复错误地结合到DNA中，导致DNA损伤和细胞死亡。尽管一些癌细胞株能够在TS抑制后积累dUTP池，但另一些则不能；这导致了一种假设，即肿瘤中dUTPase活性的升高可以防止dUTP积累导致耐药性。事实上，肿瘤标本中dUTPase的过度表达与转移性结肠癌患者对TS定向化疗的无反应有关(Ladner等人。2000)。DUTP酶是dUTP池的关键调节因子，在已被证明具有临床实用价值的途径中代表着一种有前途的替代治疗靶点。抑制dUTPase与传统药物相结合，可能为充分利用胸腺酸的生物合成作为化疗靶点提供一种新的途径。我们推测，有效的dUTPase抑制剂将增强传统TS抑制剂的疗效，并克服因瘤内dUTPase过度表达而产生的耐药性。这项建议的主要目的是下调一系列癌细胞株中dUTPase的表达，并验证抑制活性对靶向胸苷生物合成的药物疗效的影响。具体目标1：降低细胞dUTPase活性是否会使人类癌细胞对广泛使用的胸腺酸和叶酸代谢抑制剂敏感？具体目的1研究dUTPase下调对抑制胸苷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dUTPase活性下调，并提供了证明我们有能力利用这一技术的初步数据。特定目标2：TS抑制剂和dUTPase活性降低之间的协同作用是否与尿嘧啶误掺入的生化终点相关？机制分析将被用来评估尿嘧啶错结合途径下的生化要点。

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.