BIOCHEMICAL MECHANISMS OF DRUG RESISTANCE IN HIV-1 RT

HIV-1 RT 耐药的生化机制

• 批准号: 6341656
• 负责人: WALTER A SCOTT
• 金额: $ 29.39万
• 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/6341656
• 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.

3‘-叠氮胸苷(AZT)等链末端核苷类似物在HIV-1感染患者治疗中的应用受到治疗过程中耐药突变病毒选择的限制。本实验室最近描述了HIV-1逆转录酶(RT)的一种新的核苷酸依赖的焦磷分解相关活性(NPP活性)，该活性能够在生理浓度的核糖核苷三磷酸存在下从不断增长的DNA链的3‘末端去除链终止残基，并且在耐AZT的HIV中升高。NPP活性被下一个互补dNTP的生理水平所抑制，该dNTP与RT和链终止的引物/模板形成一个死端复合体。有人建议进行研究，以检验这一假设，即NPP活性及其被下一个互补dNTP的抑制是决定链末端核苷酸通过HIV-1 RT抑制DNA合成能力的主要因素。具体目的：(I)确定NPP活性在具有临床和/或机制重要性的链终止核苷酸抑制依赖RT的DNA合成中的作用；(Ii)评估引物末端、下一个互补dNTP和突变型或野生型HIV-1RT在NPP活性中的作用以及dNTPs对其抑制作用；(Iii)鉴定和/或设计抑制NPP活性的新化合物并阐明其抑制机制；建立一种灵敏的检测NPP活性产物的方法，并利用该方法监测感染HIV-1突变体的细胞中的NPP活性。HIV-1逆转录酶的NPP活性可能是决定核苷类似物治疗HIV-1感染和艾滋病疗效的主要因素。拟议的研究可能导致改进针对艾滋病毒和艾滋病的抗逆转录病毒治疗战略，并合理开发针对这一活动的新药。