Structural basis for drug resistance in HIV and FIV PRs

HIV 和 FIV PR 耐药的结构基础

• 批准号: 7860457
• 负责人: John H Elder
• 金额: $ 47.48万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-05 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7860457
• 关键词: Amino Acids; Aspartic Endopeptidases; Bacteriophages; Binding; Biological Assay; Calorimetry; Cells; Chemicals; Chimera organism; Comparative Study; Development; Distal; Drug resistance; Enzymes; Evaluation; Evolution; FIV protease; Family Felidae; Feline Immunodeficiency Virus; Gagging; Generations; Goals; HIV; HIV Protease; HIV Protease Inhibitors; HIV drug resistance; HIV-1; HIV-1 protease; Highly Active Antiretroviral Therapy; Individual; Infection; Investigation; Laboratories; Lead; Length; Libraries; Measures; Modeling; Molecular; Molecular Target; Monitor; Mutation; Patients; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Polyproteins; Process; Protease Inhibitor; Relative (related person); Research; Resistance; Resistance development; Role; Site; Specificity; Structure; Subfamily lentivirinae; Substrate Specificity; Time; Titrations; Treatment Efficacy; Variant; Viral; Viral Physiology; Virus; X-Ray Crystallography; base; drug development; drug sensitivity; fitness; improved; inhibitor/antagonist; insight; mutant; novel; pol Gene Products; pressure; research study; success; therapy development; tissue culture

The proposed research involves comparative studies using both FIV and HIV to investigate drug resistance development and substrate specificity, defining the time-course and structural basis for resistance development with the ultimate goal of providing structural information relevant to developing broad-based inhibitors. FIVs encoding chimeric protease (PR) with mutations that impart drug sensitivities and substrate specificities similar to HIV-1 PR will be generated for analysis against a panel of specific peptide substrates and phage libraries and for ex vivo infections. Progress of infections will be monitored to examine the molecular course of drug sensitivity/resistance development under single and multiple drug selections. Cleavage of peptide substrates for HIV by chimeric PRs will be used to identify residues associated with HIV substrate specificity, which previous studies have shown are not the major residues involved in resistance to current drugs. In addition, the ex vivo evolution of HIV PR in the presence and absence of PI will be assessed and information gained will inform the FIV studies. The Aims are to: 1) Prepare a battery of infectious FIV encoding "HIVinized" PR molecules in which multiple residues of FIV PR have been substituted with the equivalent residue of HIV PR. Phenotypic, genotypic, and structural analyses will be performed on virus progeny and substrate specificity changes using an ex vivo Gag processing assay will be assessed; 2) Continue to generate a panel of PR resistance mutants using novel PR inhibitors. Ex vivo competition experiments between wild type and drug-resistant HIVs will be carried out as a measure of relative viral fitness. In turn, these HIV PRs will identify additional residues to alter in FIV PR for chimeric PR studies in Aim 1. Lastly, structural evaluation of selected FIV and HIV drug-resistant PRs will be undertaken in order to assess loss of protease inhibitor (PI) potency and altered enzyme function of fully resistant PRs. Findings from these studies will provide insights into the functional plasticity of PR in regard to residue changes leading to resistance and alteration of viral function. ?? ?? ?? ?? ARRA Request: 1 R01 AI081585-01A2 Elder, John H.

拟议的研究涉及使用 FIV 和 HIV 进行比较研究，以调查耐药性的发展和底物特异性，确定耐药性发展的时间过程和结构基础，最终目标是提供与开发广泛的抑制剂相关的结构信息。编码嵌合蛋白酶 (PR) 的 FIV 具有赋予药物敏感性和类似于 HIV-1 PR 的底物特异性的突变，将生成用于针对一组特定肽底物和噬菌体库进行分析以及离体感染。将监测感染的进展，以检查单一和多种药物选择下药物敏感性/耐药性发展的分子过程。嵌合 PR 对 HIV 肽底物的切割将用于鉴定与 HIV 底物特异性相关的残基，先前的研究表明，这些残基并不是与现有药物耐药性相关的主要残基。此外，将评估在存在或不存在 PI 的情况下 HIV PR 的离体进化，获得的信息将为 FIV 研究提供信息。目的是： 1) 制备一组编码“HIVinized”PR 分子的传染性 FIV，其中 FIV PR 的多个残基已被 HIV PR 的等效残基取代。将使用离体 Gag 处理测定对病毒后代进行表型、基因型和结构分析，并评估底物特异性变化； 2) 使用新型 PR 抑制剂继续产生一组 PR 抗性突变体。野生型和耐药艾滋病毒之间的离体竞争实验将作为相对病毒适应性的衡量标准。反过来，这些 HIV PR 将识别 FIV PR 中需要改变的其他残基，用于目标 1 中的嵌合 PR 研究。最后，将对选定的 FIV 和 HIV 耐药 PR 进行结构评估，以评估蛋白酶抑制剂 (PI) 的损失完全耐药 PR 的效力和改变的酶功能。这些研究的结果将深入了解 PR 在导致耐药性和病毒功能改变的残基变化方面的功能可塑性。 ?? ?? ?? ?? ARRA 请求：1 R01 AI081585-01A2 长老，约翰 H.