STRUCTURE-BASED MECHANISMS FOR RESISTANCE TO PROTEASE INHIBITORS

项目4的目标是识别和表征蛋白酶(PR)和PR外的Gag-Pol突变 有助于对PR抑制剂(PI)的耐药性发展。我们项目过去的研究主要集中在 关于公关内部导致耐药性的变化。然而，最近的发现表明，更多的 突变出现在Gag-Pol中，补充PR中的耐药突变。这些次要的 突变既发生在自然裂解部位，也发生在Gag-Pol的其他区域。这些发现限制了实用程序 只研究PR中的耐药性突变以指导新的PI的发展并暗示非蛋白水解酶 突变必须被考虑在内。因此，我们将研究PI-定向进化 组织培养和猪瘟患者序贯标本中耐药发展的研究 治疗。我们提出了四个具体目标来完成我们的目标：1)识别和确定亲属 极端条件下PR和Gag-Pol基因突变在PR外耐药中的作用 组织培养中“乒乓球”酶抑制剂的选择。2)表征来自以下位置的连续病毒样本 在对临床核心提供的临床数据集的分析中确定的患者对 精选的几类蛋白酶抑制剂和在蛋白酶期间被“乒乓球”处理的患者 抑制剂治疗。3)根据AIMS 1和AIMS 2产生的蛋白酶突变体将用于选择和 与项目3合作产生的新的抑制剂和独特的PR也将用作目标 4)结合结构和蛋白质表达的片段筛选分析 我们将对突变的PR和Gag-Pol的靶区进行结构研究，以建立一个 在AIMS 1和2中确定的PI抗性的分子基础。拟议的研究将大大拓宽 我们过去15年进行的研究的范围，将提供宝贵的信息 与了解和应对PR和Gag-Pol耐药性发展有关的研究 目前和未来的艾滋病毒药物疗法。 外行语言：对艾滋病毒药物的耐药性是世界范围内的一个主要问题。我们的研究针对的是 确定艾滋病毒耐药性是如何发生的，并利用这一知识开发出更好的药物， 控制耐药-艾滋病毒和那些更难发展的艾滋病毒耐药性。

Project 4 goals are to identify and characterize mutations in protease (PR) and Gag-Pol outside of PR that contribute to resistance development against PR inhibitors (PI). Past studies by our program have focused on changes within PR that impart drug resistance. However, recent findings indicate that additional mutations arise in Gag-Pol that complement drug resistance mutations within PR. These secondary mutations occur both within natural cleavage sites and other areas of Gag-Pol. These findings limit the utility of studying resistance mutations solely within PR to direct development of new Pis and imply that nonprotease mutations must be taken into consideration. Therefore, we will investigate the Pi-directed evolution of resistance development in both tissue culture and in sequential samples from patients undergoing PI treatment. We propose four Specific Aims to complete our goals: 1) Identify and determine the relative contributions to drug resistance of mutations in PR and Gag-Pol outside of PR that arise under extreme "ping-pong" protease inhibitor selection in tissue culture. 2) Characterize sequential virus samples from patients identified in the analyses of the clinical data set provided by the Clinical Core that were resistant to selected classes of protease inhibitors and from patients that have been "ping-ponged" during protease inhibitor treatment. 3) Protease mutants generated under Aims 1 and 2 will be used for selection and generation of new inhibitors in collaboration with Project 3 and unique PRs will also be used as targets for fragment screening analyses under Project 2. 4) In collaboration with the Structural & Protein Expression Core, we will perform structural studies on mutant PRs and targeted regions of Gag-Pol to establish a molecular basis for PI resistance identified in Aims 1 & 2. The proposed studies will substantially broaden the scope of our previous studies performed over the past 15 years and will provide invaluable information relevant to understanding and responding to resistance development of PR and Gag-Pol observed using current and future HIV drug therapies. Lay Language: Resistance to HIV drugs is a major problem world-wide. Our studies are directed at determining how drug resistance to HIV occurs and to use this knowledge to develop better drugs that control drug-resistant-HIV and that are more difficult for HIV resistance development.