BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE IN HIV-1 RT

HIV-1 RT 耐药的生化机制

• 批准号: 6080195
• 负责人: WALTER A SCOTT
• 金额: $ 28.55万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-15 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6080195
• 关键词: RNA directed DNA polymerase; antiAIDS agent; deoxyribonucleoside triphosphate; drug design /synthesis /production; drug resistance; enzyme activity; enzyme mechanism; human immunodeficiency virus 1; method development; molecular cloning; protein purification; protein sequence; zidovudine

The use of chain-terminating nucleoside analogues such as 3'-azidothymidine (AZT) in the treatment of HIV-1 infected patients is limited by the selection of drug-resistance mutant virus during therapy. This laboratory has recently described a novel nucleotide-dependent, pyrophosphorolysis-related activity (NPP activity) of HIV-1 reverse transcriptase (RT) that is capable of removing a chain-terminating residue from the 3'- terminus of the growing DNA chain in the presence of physiological concentrations of ribonucleoside triphosphates and is elevated in AZT-resistant HIV. NPP activity is inhibited by physiological levels of the next complementary dNTP which forms a dead-end complex with RT and chain-terminated primer/template. Research is proposed to test the hypothesis that NPP activity and its inhibition by the next complementary dNTP are major factors in determining the ability of chain-terminating nucleotides to inhibit DNA synthesis by HIV-1 RT. Specific Aims: (i) To determine the role of NPP activity in the inhibition of RT-dependent DNA synthesis by chain-terminating nucleotides that are of clinical and/or mechanistic importance; (ii) To evaluate the role of the primer terminus, the next complementary dNTP, and mutant or wild type HIV-1 RT in NPP activity and its inhibition by dNTPs; (iii) To identify and/or design new compounds that inhibit NPP activity and to elucidate the mechanism of their inhibition; and (iv) To develop a sensitive method to detect the products of NPP activity and to use this assay to monitor NPP activity in cells infected with mutants of HIV-1. The NPP activity of HIV-1 RT may be a major factor determining the effectiveness of nucleoside analogues in the treatment of HIV-1 infection and AIDS. The proposed research may lead to improved strategies for antiretroviral therapy against HIV and AIDS and the rational development of new drugs that target this activity.

在治疗期间选择药物耐药突变病毒的限制了链终止的核苷类似物，例如3'-氮杂酪氨酸（AZT）在治疗HIV-1感染患者中的使用受到限制。 该实验室最近描述了HIV-1逆转录酶（RT）的新型核苷酸依赖性，与焦磷酸相关的活性（NPP活性），该活性能够从生理浓度的三核固定浓度的三链中，能够从生长的DNA链的3'-末端中取出链末端的残基。 下一个互补DNTP的生理水平抑制了NPP活性，该互补DNTP与RT和链端启动/模板形成死端复合物。提出了研究来检验下一个互补DNTP的NPP活性及其抑制作用的假设是确定链末端核苷酸抑制HIV-1 RT抑制DNA合成的能力的主要因素。具体目的：（i）确定NPP活性在抑制RT依赖性DNA合成中的作用，该链末端的核苷酸具有临床和/或机械意义； （ii）评估引物末端的作用，下一个补充DNTP以及突变或野生型HIV-1 RT在NPP活性中及其抑制DNTP的作用； （iii）识别和/或设计抑制NPP活性并阐明其抑制机制的新化合物； （iv）开发一种敏感方法来检测NPP活性的产物，并使用该测定法监测感染HIV-1突变体的细胞中的NPP活性。 HIV-1 RT的NPP活性可能是确定核苷类似物在HIV-1感染和AIDS治疗中的有效性的主要因素。 拟议的研究可能导致改善针对艾滋病毒和艾滋病的抗逆转录病毒疗法的策略，以及针对该活动的新药的合理发展。