Engineering enzymes for anti-tumor suicide gene therapy

用于抗肿瘤自杀基因治疗的工程酶

• 批准号: 7054653
• 负责人: BARRY L. STODDARD
• 金额: $ 31.24万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7054653
• 关键词: X ray crystallography; aminohydrolases; antineoplastics; athymic mouse; bacterial proteins; cell line; cytotoxicity; dCMP deaminase; deoxycytidine; disease /disorder model; drug metabolism; drug screening /evaluation; enzyme activity; enzyme substrate; fluorouracil; fungal proteins; gemcitabine; gene complementation; gene therapy; genetic screening; neoplastic cell; prodrugs; protein purification; site directed mutagenesis; structural biology

DESCRIPTION (provided by applicant): Prodrug gene therapy is a therapeutic strategy in which tumor cells are transfected with a 'suicide' gene that encodes a metabolic enzyme which is capable of converting a nontoxic prodrug into a potent cytotoxin. Such a method allows selective eradication of tumor cells while sparing normal tissue from significant cell killing. The effectiveness of this strategy is dependent on a bystander effect in which untransfected tumor cells are killed through active or passive transport of the cytotoxic enzyme product. Several enzyme/prodrug combinations are under active investigation, demonstrating effectiveness in both tissue culture and animal models. However, the combination of low transfection efficiencies and poor turnover of prodrug substrates limit the efficiency of cell killing in the tumor. In order to improve such therapies, enzyme variants must be selected and engineered for enhanced turnover of the prodrug substrate. In this proposal, a collaboration of two laboratories propose to optimize the nucleoside salvage enzymes cytosine deaminase and deoxycytidine kinase for prodrug suicide gene therapy, using a combination of structural biology and directed evolution screens, and to test the efficacy of enzyme variants in cell line and animal models. Cytosine deaminase (CD) catalyzes the deamination of cytosine to uracil and ammonia. Cytosine deaminase is found in bacteria and fungi but is not present in mammalian cells. Cells expressing CD are sensitive to the nucleoside analog, 5-fluorocytosine. Due to the enzymatic conversion of 5FC to 5-fluorouracil (5FU). This compound and its deoxyribonucleoside, fluorodeoxyuridine (FUdR), are potent inhibitors of DNA synthesis and RNA function and are widely used in cancer treatment. In contrast, deoxycytidine kinase (dCK) generates cytidine-monophosphate from cytidine nucleoside, and also activates the antineoplastic agents gemcitabine and cytarabine. The specific aims for this project are: (1) Determine the structure of bacterial CD, yeast CD and human dCK. (2) Perform structure-based mutagenesis and genetic screens to isolate enzyme variants with enhanced binding and turnover of prodrug substrates. This aim will exploit crystallographic information both to direct the mutagenesis of specific regions of the enzyme, and to directly visualize the structural basis of enhanced prodrug. activation by selected enzyme variants (3) Test enzyme variants for tumor cell killing in established tumor model systems, using cell lines and animal models.

描述（由申请人提供）：前药基因治疗是一种治疗策略，其中肿瘤细胞用“自杀”基因转染，该基因编码代谢酶，该酶能够将无毒药物转化为有效的细胞毒素。这种方法允许选择性地消除肿瘤细胞，同时避免正常组织重大细胞杀伤。该策略的有效性取决于旁观者效应，在旁观者的效果下，未转染的肿瘤细胞通过细胞毒性酶产物的主动或被动转运杀死。几种酶/前药组合正在积极研究中，证明了组织培养和动物模型的有效性。但是，低转染效率和前药底物营业额差的结合限制了肿瘤中细胞杀死的效率。为了改善这种疗法，必须选择并设计酶变体以增强前药底物的营业额。在这项建议中，两家实验室的合作提议优化核苷酶透明酶胞嘧啶脱氨酶脱氨酶和脱氧胞苷激酶，以使用结构生物学和定向进化筛选的结合，用于前药自杀基因疗法，并测试Cell Line和动物模型的Enzyme Variants和动物模型的效率。胞嘧啶脱氨酶（CD）催化胞嘧啶对尿嘧啶和氨的脱氨基。胞嘧啶脱氨酶在细菌和真菌中发现，但在哺乳动物细胞中不存在。表达CD的细胞对核苷类似物，5-氟环胞嘧啶敏感。由于5FC向5-Fluorouracil（5FU）的酶促转化。该化合物及其脱氧核糖核苷，氟脱氧尿苷（FUDR）是DNA合成和RNA功能的有效抑制剂，并广泛用于癌症治疗中。相比之下，脱氧胞苷激酶（DCK）从胞丁核苷产生胞丁磷酸，也激活抗肿瘤剂吉西他滨和胞滨。该项目的具体目的是：（1）确定细菌CD，酵母CD和人类DCK的结构。 （2）执行基于结构的诱变和遗传筛选，以分离酶变体，并具有增强的前药底物结合和周转率。这个目标将利用晶体学信息来指导酶的特定区域的诱变，并直接可视化增强前药的结构基础。使用细胞系和动物模型在已建立的肿瘤模型系统中，通过选定的酶变体（3）测试酶杀死肿瘤细胞杀死的酶变体的激活。