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）：在与现有和新出现的病毒感染和癌症作斗争时，核苷类似物（NAS）代表了药用武器库中的突出且高度有效的武器。作为膜渗透核苷前药的施用，化合物需要脱氧核苷激酶（DNKS）和脱氧核苷酸激酶（DNPKS）在其生物活性的三磷酸同行者中进行细胞内激活。近年来，DNK和DNPK在寻求新的，更有效的抗病毒药和抗癌药物方面发挥了关键作用。长期暴露，细胞毒性中间体的积累以及大部分的新前药体内的失败与与磷酸化级联有关的问题有关。拟议的研究计划着重于哺乳动物和细菌DNK和DNPK的工程，定制催化剂，以最佳的NAS特异性，以最大程度地提高癌症和抗病毒治疗方面的前药效率。采用一种名为Scratchy的新型，同源性独立的组合技术，使我们能够从DNK/DNPK家族中发现的高序列多样性的父母产生大量的混合构造。随后通过遗传选择和高通量筛选选择功能性杂种候选物。我们将1）证明，蛋白质片段在结构同源蛋白之间交换可以产生表现出所需功能的杂化酶，2）通过与原始的基于细胞的测定中的前药进行验证杂交激酶的性能，以及3）开发一个框架以研究DNK和DNK和DNK和DNPS的结构函数关系。这项工作的具体目的是：1）鉴定具有改进的NA特异性的工程DNK，2）生成具有多步磷酸化的新型催化剂，3）以改善基于结构的蛋白质工程的多功能性，以及4）评估既定的NAS NAS NAS NAS Intibore and Nempe nas Intipro and Vivo。这些研究的结果将对病毒疾病和癌症治疗产生深远的影响，从而通过促进其磷酸化而独立于细胞DNK激活来影响现有前药以及潜在的候选药物。最后，混合蛋白的结构多样性将成为基本结构功能研究的丰富来源。