MECHANISMS OF NUCLEOSIDES INDUCED TOXICITY AND ACTIVITY

核苷诱导毒性和活性的机制

• 批准号: 2330375
• 负责人: JEAN-PIERRE C SOMMADOSSI
• 金额: $ 20.51万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2330375
• 关键词: 2'3' dideoxynucleoside; RNA directed DNA polymerase; antiAIDS agent; cell line; drug adverse effect; drug metabolism; enzyme activity; enzyme inhibitors; human immunodeficiency virus; human tissue; nucleoside analog; protein kinase; protein purification; site directed mutagenesis; stereoisomer

DESCRIPTION (Adapted from the Investigator's Abstract): The long term objectives of this renewal application are to investigate and extend ongoing studies on the cellular and molecular mechanism(s) by which nucleoside analogs selectively target the reverse transcriptase of the human immunodeficiency virus (HIV). Extensive studies on the interaction of these drugs with the host cell have been shown in the past funding period to be critical in our understanding of the underlying mechanism(s) of drug action and toxicity. The synthesis of novel L-purine nucleoside analogs and the PI's recent discovery that a new series of L-dideoxyadenosine analogs are potent inhibitors of HIV and HBV replication demonstrates for the first time that development of L-purine derivative should also be vigorously pursued. These data provide a strong rationale for elucidation of their mechanisms of action and metabolism in target cells of these viral infections. This project includes three major aims: (I) Identification of the metabolic pathways and mechanism of action of L-purine analogs with particular emphasis on L-dideoxyadenosine derivatives. This proposal will evaluate the cellular metabolism (anabolism and catabolism) and compartmentalization of L-ddA, L-d4A and derivative in established cell lines and in human primary cells which are infectable by HIV, including lymphocytes and monocyte-macrophages. Selective inhibitors and mutant genetically defective (dCK-, AK-, etc.) cells will be used to elucidate activating kinase enzymes involved in the activation processes of these nucleosides. Nucleoside kinases responsible for nucleoside anabolism will be purified and kinetic constants for the various nucleosides will be determined. Effect of structural alterations on substrate specificities targeted toward maximization of antiviral activity will be assessed. The impact of stereoisomerism of the beta-enantomers of purine analogs on their cellular metabolism as well as their interaction with the antiviral target and/or hypothesized toxicity sites will be examined, including the assessment of potential combination therapies through metabolic interactions. (II) Evaluation of rationally designed pronucleotides incorporating enzyme-mediated bioreversible protection groups allowing the direct intracellular delivery of b-L-nucleoside-5'-monophosphate. (III) Investigations of the potential for HIV-1 resistance development to L-purine nucleoside analogs by phenotypic and genotypic characterization; confirmation of the role of specific mutations by site-specific mutational analysis; examination of potential cross-resistance to clinically approved nucleoside analogs; impact of L-purine nucleoside resistant mutation of HIV-RT on substrate/inhibitor recognition by beta-L-5'-triphosphate derivatives.

描述（根据调查员的摘要改编）：长期 此更新应用程序的目标是调查和扩展正在进行的 核苷的细胞和分子机制的研究 类似物选择性地靶向人类的逆转录酶 免疫缺陷病毒（HIV）。 关于这些相互作用的广泛研究 在过去的资金期间已经显示了带有宿主细胞的药物 我们对毒品作用的基本机制的理解至关重要 和毒性。 新型L-铅核苷类似物的合成和 Pi最近发现的新系列L-脱氧腺苷类似物是 HIV和HBV复制的有效抑制剂首次证明 L-吡啶衍生物的发展也应大力追求。 这些数据为阐明其机制的机制提供了有力的理由 这些病毒感染的靶细胞的作用和代谢。 这 项目包括三个主要目的：（i）代谢的识别 特定的L-钢铁类似物的途径和作用机理 强调L-脱氧腺苷衍生物。 该建议将评估 细胞代谢（代谢和分解代谢）和分隔 在已建立的细胞系和人类原理中的L-DDA，L-D4A和衍生物 通过HIV感染的细胞，包括淋巴细胞和 单核细胞巨噬细胞。 选择性抑制剂和突变体遗传缺陷 （DCK-，AK-等）将使用细胞阐明激活激活酶 参与这些核苷的激活过程。 核苷 负责核苷代谢的激酶将被纯化和动力学 将确定各种核苷的常数。 效果 针对底物特异性的结构改变 将评估抗病毒活性的最大化。 的影响 嘌呤类似物在其细胞上的beta beta beta型的立体异构主义 代谢以及与抗病毒靶的相互作用和/或 将检查假设的毒性部位，包括评估 潜在的组合疗法通过代谢相互作用。 （ii） 评估合理设计的核苷酸合并 酶介导的生物可逆保护组允许直接 B-L-核苷-5'单磷酸盐的细胞内递送。 （iii） 研究HIV-1耐药性向L-purine的潜力的研究 通过表型和基因型表征进行核苷类似物； 通过位点特异性突变确认特定突变的作用 分析;检查潜在的交叉耐药性到临床批准 核苷类似物； L-吡啶核苷抗性突变的影响 HIV-RT对β-L-5'-3-磷酸盐识别的底物/抑制剂识别 衍生物。