Structural Biology of Enzymes Activating Prodrugs

酶激活前药的结构生物学

• 批准号: 7002495
• 负责人: ARNON LAVIE
• 金额: $ 23.33万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7002495
• 关键词: X ray crystallography; alcohol phosphotransferase; antineoplastics; antiviral agents; chemical kinetics; computer simulation; conformation; drug discovery /isolation; drug metabolism; enzyme mechanism; enzyme structure; enzyme substrate complex; fluorescent dye /probe; gene therapy; gram negative bacteria; human genetic material tag; intermolecular interaction; molecular site; nucleoside analog; nucleoside phosphate kinase; phosphorylation; prodrugs; site directed mutagenesis; structural biology; thymidylate kinase

DESCRIPTION (provided by applicant): The efficacy of nucleoside analog (NA) prodrugs in anticancer and antiviral treatments is often impaired by the slow intracellular activation of these drugs. NAs are administered as the uncharged nucleoside to achieve effective intracellular concentrations. Once inside the cell, NAs require conversion to their triphosphorylated form to become pharmacologically active. The sequential three-step phosphorylation is carried out by human cellular kinases, with the first step, catalyzed by nucleoside kinases, most often being rate limiting. For example, both AZT and d4T require conversion into their monophosphate form by human thymidine kinase, which for d4T is the rate-limiting step in its activation. To address the problem of inefficient NA activation we propose to structurally and kinetically characterize human thymidine kinases. Vertebrates have two different thymidine kinases, a cytosolic form (hTK1) and a mitochondrial form (hTK2). Activity of hTK1 is high in proliferating cells and peaks during the S-phase of the cell cycle, while hTK2 is constitutively expressed at low levels. To understand the molecular determinants of substrate specificity, enzymatic mechanism, and regulatory mechanisms of human thymidine kinases we propose to: (1) Solve the crystal structures of hTK1 in complex with substrates and NA prodrugs. (2) Delineate the regulatory mechanisms controlling hTK1 activity. (3) Characterize the determinants of substrate specificity and investigate the catalytic mechanism of hTK2. (4) Delineate the structural reasons that confer high d4T-phosphorylating activity of the homologous T. maritima thymidine kinase. (5) Based on the results from the previous aims, design and produce mutants of the human enzymes that possess higher activity and improved specificity for clinically relevant prodrugs. Together, the proposed work will dramatically increase our understanding of these important enzymes. In addition, the results will improve our abilities to develop NAs that are selectively activated by TK1 and not by TK2, a major goal since activation by TK2 often results in toxic side effects. A long-term goal of this project is to engineer enzymes that possess improved prodrug activation efficiencies to be employed in antiviral and anticancer therapies.

描述（由申请人提供）：核苷类似物（NA）前药在抗癌和抗病毒治疗中的功效常常因这些药物的缓慢细胞内激活而受到损害。 NA 作为不带电荷的核苷施用以达到有效的细胞内浓度。 一旦进入细胞，NA 需要转化为三磷酸化形式才能具有药理活性。 连续的三步磷酸化是由人类细胞激酶进行的，第一步由核苷激酶催化，最常见的是限速。 例如，AZT 和 d4T 都需要通过人胸苷激酶转化为单磷酸形式，这对于 d4T 来说是其激活的限速步骤。 为了解决 NA 激活效率低下的问题，我们建议对人胸苷激酶进行结构和动力学表征。 脊椎动物有两种不同的胸苷激酶：胞质形式 (hTK1) 和线粒体形式 (hTK2)。 hTK1 的活性在增殖细胞中较高，并在细胞周期的 S 期达到峰值，而 hTK2 则持续低水平表达。 为了了解人胸苷激酶底物特异性、酶促机制和调节机制的分子决定因素，我们建议：（1）解析hTK1与底物和NA前药复合物的晶体结构。 (2) 描述控制 hTK1 活性的调控机制。 (3)表征底物特异性的决定因素并研究hTK2的催化机制。 (4) 描述赋予同源 T. maritima 胸苷激酶高 d4T 磷酸化活性的结构原因。 (5) 基于先前目标的结果，设计和生产具有更高活性和临床相关前药特异性的人类酶突变体。 总之，拟议的工作将极大地增加我们对这些重要酶的理解。 此外，这些结果将提高我们开发由 TK1 而不是 TK2 选择性激活的 NA 的能力，这是一个主要目标，因为 TK2 激活通常会导致毒副作用。 该项目的长期目标是设计具有改进的前药激活效率的酶，用于抗病毒和抗癌治疗。