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）：这些药物的细胞内激活缓慢会损害核苷类似物（NA）前药在抗癌和抗病毒药物治疗中的疗效。 NAS被用作未加成的核苷以实现有效的细胞内浓度。 一旦进入细胞，NAS就需要转换为三磷酸化形式才能成为药理活性。 顺序三步磷酸化是由人类细胞激酶进行的，第一步是由核苷激酶催化的，最常见于速率限制。 例如，AZT和D4T都需要通过人胸苷激酶转化为单磷酸形式，这是D4T的激活速率限制步骤。 为了解决无效NA激活的问题，我们建议在结构和动力学上表征人胸苷激酶。 脊椎动物具有两种不同的胸苷激酶，一种胞质形式（HTK1）和线粒体形式（HTK2）。 在细胞周期的S期间，HTK1的活性在增殖细胞和峰值上很高，而HTK2则在低水平的组成型表达。 为了了解人胸苷激酶的底物特异性，酶机制和调节机制的分子决定因素，我们建议：（1）与底物和Na前药中的复合物中的HTK1晶体结构求解。 （2）描述控制HTK1活性的调节机制。 （3）表征底物特异性的决定因素并研究HTK2的催化机理。 （4）描述赋予同源t. maritima胸苷激酶的高D4T磷酸化活性的结构原因。 （5）基于先前目标的结果，设计和产生人类酶的突变体，这些突变体具有较高的活性和提高临床相关前药的特异性。 共同提议的工作将大大增加我们对这些重要酶的理解。 此外，结果将提高我们开发由TK1选择性激活而不是TK2的NA的能力，这是一个主要目标，因为TK2激活通常会导致毒性副作用。 该项目的一个长期目标是设计具有改进的前药激活效率的酶，可用于抗病毒和抗癌疗法。