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（改编自研究者摘要）：长期 此更新申请的目的是调查并扩展正在进行的 研究核苷发挥作用的细胞和分子机制 类似物选择性地靶向人类的逆转录酶 免疫缺陷病毒（HIV）。 对这些相互作用的广泛研究 在过去的资助期内，具有宿主细胞的药物已被证明是 对于我们理解药物作用的潜在机制至关重要 和毒性。 新型L-嘌呤核苷类似物的合成及其 PI最近发现一系列新的L-双脱氧腺苷类似物 首次证明有效的 HIV 和 HBV 复制抑制剂 还应大力发展L-嘌呤衍生物。 这些数据为阐明其机制提供了强有力的依据。 这些病毒感染的靶细胞中的作用和代谢。 这 项目包括三个主要目标：（一）代谢的鉴定 L-嘌呤类似物的作用途径和机制 重点关注L-双脱氧腺苷衍生物。 该提案将评估 细胞代谢（合成代谢和分解代谢）和区室化 L-ddA、L-d4A 及其衍生物在已建立的细胞系和人类原代细胞中 受HIV感染的细胞，包括淋巴细胞和 单核细胞-巨噬细胞。 选择性抑制剂和遗传缺陷突变体 （dCK-、AK- 等）细胞将用于阐明激活激酶 参与这些核苷的激活过程。 核苷 负责核苷合成代谢的激酶将被纯化并具有动力学特性 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 嘌呤类似物的β-对映异构体在细胞上的立体异构现象 代谢以及它们与抗病毒靶点的相互作用和/或 将检查假设的毒性位点，包括评估 通过代谢相互作用的潜在联合疗法。 （二） 合理设计的前核苷酸整合的评估 酶介导的生物可逆保护基团允许直接 b-L-核苷-5'-单磷酸的细胞内递送。 （三） HIV-1 对 L-嘌呤产生耐药性的可能性的研究 通过表型和基因型表征确定核苷类似物； 通过位点特异性突变确认特定突变的作用 分析;检查与临床批准的潜在交叉耐药性 核苷类似物； L-嘌呤核苷抗性突变的影响 HIV-RT 通过 β-L-5'-三磷酸识别底物/抑制剂 衍生物。