SELECTIVE INHIBITION OF HCV TRANSLATION BY TETRAPEPTIDE

四肽对 HCV 翻译的选择性抑制

基本信息

批准号：
7260416
负责人：
ALEXANDER V BIRK
金额：
$ 10.8万
依托单位：
BARUCH S. BLUMBERG INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-08-01 至 2009-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7260416
关键词：
5&apos Untranslated Regions Adverse effects Arginine Binding Biochemistry and Pharmacology Cancer Activity Biological Assay Cells Chemicals Chronic DNA Digestion Elements Endoribonucleases Genetic Translation Goals Hepatitis C Hepatitis C virus Hepatocyte Human In Vitro Internal Ribosome Entry Site Length Life Cycle Stages Liver Cirrhosis Liver diseases Luciferases Lysine Mediating Northern Blotting Opioid Opioid Peptide Pancreatic ribonuclease Peptide Hydrolases Peptides Primary carcinoma of the liver cells Protein Biosynthesis RNA RNA Binding RNA Interference RNA chemical synthesis RNA replication RNase protection assay Regulation Replicon Reporter Research Resistance Reticulocytes Ribonucleases Role Sedimentation process Signal Transduction Site Structure Sucrose System Therapeutic Translation Initiation Translations Tyrosine Viral Proteins Water Western Blotting analog anti-hepatitis C base clinical application cytotoxic design improved inhibitor/antagonist lysylphenylalanine mRNA capping phenylalanylarginine success synthetic peptide ultraviolet irradiation uptake vector viral RNA

项目摘要

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) is a major cause of chronic liver disease, liver cirrhosis and hepatocellular carcinoma. Preliminary studies with a water-soluble, cell-permeable and non-cytotoxic synthetic peptide called DAPL (Dmt-D-Arg-Phe-Lys-NH2, where Dmt is 2', 6'- dimethyltyrosine) demonstrate that this peptide can selectively interact with the HCV RNA translation initiation site (HCV IRES) and inhibit HCV translation selectively in vitro. The goal of this proposal is to explore the biochemistry, pharmacology and mechanism of action of DAPL in inhibiting HCV translation, the key element of HCV infection. Three specific aims were established: (1) Studies on regulation of HCV-IRES-mediated translation by DAPL in vitro. Thus, we plan to demonstrate an effect of DAPL on HCV translation in vitro and determine the site(s) of DAPL-IRES interaction, by determining the sites of the IRES protected by DAPL from enzymatic, chemical, and physical probing. We will also determine the effect of DAPL on ribosomal assembly on HCV IRES, by determining the effect of DAPL on HCV RNA binding to ribosomal subunits and the ribosomal protection of HCV IRES from RNase digestion. (2) To examine the ability of DAPL to regulate HCV translation and replication in cells. Thus, the effect of DAPL on HCV IRES-directed translation and HCV RNA synthesis in Huh 7 and Huh-7.5 cells transfected with DNA vectors carrying reporters (luciferase) and HCV replicon, respectively will be determined with Northern and Western assays. (3) To investigate structural requirements of DAPL for selective inhibition of HCV translation. Thus, we plan to investigate the role of dimethyl-tyrosine, arginine, and different lengths terminal lysines in the regulation of HCV translation in vitro and their interaction with HCV IRES. If DAPL or related analogues can be further developed to target and inhibit HCV RNA translation, the clinical application of this kind of therapeutics might be significant.

描述（由申请人提供）：丙型肝炎病毒（HCV）是慢性肝病、肝硬化和肝细胞癌的主要原因。对称为 DAPL（Dmt-D-Arg-Phe-Lys-NH2，其中 Dmt 是 2', 6'- 二甲基酪氨酸）的水溶性、细胞渗透性和非细胞毒性合成肽的初步研究表明，该肽可以选择性地相互作用与 HCV RNA 翻译起始位点 (HCV IRES) 结合并在体外选择性抑制 HCV 翻译。本提案的目的是探讨 DAPL 抑制 HCV 翻译（HCV 感染的关键因素）的生物化学、药理学和作用机制。确定了三个具体目标：（1）研究DAPL体外调节HCV-IRES介导的翻译。因此，我们计划通过酶促、化学和物理探测确定受 DAPL 保护的 IRES 位点，从而在体外证明 DAPL 对 HCV 翻译的影响，并确定 DAPL-IRES 相互作用的位点。我们还将通过确定 DAPL 对 HCV RNA 与核糖体亚基结合的影响以及 HCV IRES 核糖体保护免受 RNase 消化的影响，确定 DAPL 对 HCV IRES 上核糖体组装的影响。 (2)考察DAPL调节细胞内HCV翻译和复制的能力。因此，DAPL对分别用携带报道分子（荧光素酶）和HCV复制子的DNA载体转染的Huh 7和Huh-7.5细胞中HCV IRES指导的翻译和HCV RNA合成的影响将通过Northern和Western测定来确定。 (3)研究DAPL选择性抑制HCV翻译的结构要求。因此，我们计划研究二甲基酪氨酸、精氨酸和不同长度的末端赖氨酸在体外调节HCV翻译中的作用以及它们与HCV IRES的相互作用。如果能够进一步开发DAPL或相关类似物来靶向和抑制HCV RNA翻译，则此类疗法的临床应用可能具有重要意义。