Improving nucleoside phosphorylation with hybrid kinases

用混合激酶改善核苷磷酸化

• 批准号: 6972945
• 负责人: STEFAN LUTZ
• 金额: $ 27.77万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6972945
• 关键词: 2' 3' dideoxynucleoside; biochemistry; biophysics; chimeric proteins; combinatorial chemistry; enzyme substrate; high throughput technology; method development; microorganism disease chemotherapy; neoplasm /cancer chemotherapy; nucleosides; phosphorylation; prodrugs; protein engineering; protein structure function

DESCRIPTION (provided by applicant): In battling existing and newly emerging viral infections and cancer, nucleoside analogues (NAs) represent a prominent and highly potent weapon in the medicinal arsenal. Administered as membrane-penetrating nucleosidic prodrugs, the compounds require intracellular activation by deoxynucleoside kinases (dNKs) and deoxynucleotide kinases (dNPKs) into their bioactive triphosphate anabolites. In recent years, dNKs and dNPKs have taken on a key role in the quest for new and more potent antiviral and anticancer drugs. Emerging resistance to NAs upon long-term exposure, accumulation of cytotoxic intermediates, and failure of a large percentage of new prodrugs in vivo has been linked to problems associated with the phosphorylation cascade. The proposed research program focuses on the engineering of mammalian and bacterial dNKs and dNPKs, tailoring catalysts for optimal specificity toward NAs to maximize prodrug efficiency upon codelivery in cancer and antiviral therapy. Employing a novel, homology- independent combinatorial technique named SCRATCHY enables us to produce vast numbers of hybrid constructs from parents with high sequence diversity as found in the dNK/dNPK family. Functional hybrid candidates are subsequently selected by genetic selection and high-throughput screening. We will 1) Demonstrate that protein fragment swapping between structure-homologous proteins can generate hybrid enzymes that exhibit desired function, 2) Validate the performance of hybrid kinases through co-administration with prodrugs in cell-based assays, and 3) Develop a framework to study the structure-function relationship of dNKs and dNPKs. The specific aims of this work are: 1) To identify engineered dNKs with improved NA specificity, 2) To generate novel catalysts that perform multistep phosphorylation, 3) To improve the versatility of structure-based protein engineering, and 4) To evaluate functional hybrid enzymes on established and novel NAs in vitro and in vivo. The results from these studies will have far-reaching implications on viral disease and cancer treatment, affecting existing prodrugs as well as potential drug candidates by facilitating their phosphorylation independent of cellular dNK activation. Finally, the structural diversity of the hybrid proteins will be a rich source for fundamental structure-function studies.

描述（由申请人提供）：在对抗现有和新出现的病毒感染和癌症时，核苷类似物（NA）代表了医学武器库中的一种突出且高效的武器。这些化合物作为膜穿透核苷前药施用，需要通过脱氧核苷激酶 (dNK) 和脱氧核苷酸激酶 (dNPK) 在细胞内激活为其生物活性三磷酸合成代谢物。近年来，dNK 和 dNPK 在寻找新的、更有效的抗病毒和抗癌药物中发挥了关键作用。长期暴露后出现的对 NA 的耐药性、细胞毒性中间体的积累以及体内大部分新前药的失效都与磷酸化级联相关的问题有关。拟议的研究计划重点关注哺乳动物和细菌 dNK 和 dNPK 的工程设计，定制催化剂以实现对 NA 的最佳特异性，从而最大限度地提高癌症和抗病毒治疗中共递送时的前药效率。采用一种名为 SCRATCHY 的新型同源独立组合技术，使我们能够从具有高序列多样性的亲本产生大量杂交构建体，如 dNK/dNPK 家族中发现的那样。随后通过遗传选择和高通量筛选来选择功能性杂交候选物。我们将 1) 证明结构同源蛋白质之间的蛋白质片段交换可以产生具有所需功能的杂合酶，2) 通过在基于细胞的测定中与前药共同施用来验证杂合激酶的性能，以及 3) 开发一个框架研究 dNK 和 dNPK 的结构与功能关系。这项工作的具体目标是：1) 鉴定具有改进的 NA 特异性的工程化 dNK，2) 生成执行多步磷酸化的新型催化剂，3) 提高基于结构的蛋白质工程的多功能性，4) 评估功能杂化体外和体内已建立的和新型 NA 上的酶。这些研究的结果将对病毒性疾病和癌症治疗产生深远的影响，通过促进独立于细胞 dNK 激活的磷酸化来影响现有的前药以及潜在的候选药物。最后，杂合蛋白的结构多样性将成为基础结构功能研究的丰富来源。