Elucidating Molecular Mechanisms of Drug Resistance in HIV-1 Protease

描述(申请人提供)：HIV-1感染是一种世界性的流行病。尽管目前的药物疗法在延长患者生命方面是有效的，但感染仍在继续蔓延，特别是在非发达国家，而且耐药性的出现限制了许多蛋白酶抑制剂的有效性。HIV-1蛋白水解酶(HIV-1PR)附属突变的进化在恢复酶功能的同时使其对多种抑制剂产生交叉耐药的机制是本研究的重点。我们提出了一种新的机制，它唤起了蛋白质构象采样，作为解释次级突变如何完成这一任务的分子基础。具体而言，我们假设突变在四种名义状态之间的组合模式会改变HIV-1PR的构象采样；即关闭状态、半开放状态、卷曲/折叠状态和完全开放状态，从而(1)当它们组合在一起以将半开放构象稳定到在天然酶(例如抑制物朴素序列)中观察到的百分比时，酶功能可由次级突变恢复；(2)当突变组合以稳定“类开放”状态(如完全开放和卷曲/折叠构象)时，就会出现交叉耐药性，同时伴随着破坏关闭状态人口的稳定。正是通过脉冲电子顺磁光谱学的创新应用，我们的实验室在过去五年中开创了这一应用，我们测量了HIV-1PR的构象样本集合。在这项建议的目标内，我们将在HIV-1PR构象样本集合的变化与酶参数、抑制常数、抑制剂敏感性和许多HIV-1PR变体的病毒适合性之间建立关联。从这些结果中，如果我们的假设是正确的，我们将提供分子水平的理解，以了解继发性突变是如何引起它们对HIV-1PR的影响的。此外，这些研究还将为解释为什么在非B亚型中针对蛋白酶抑制剂的二次突变进化模式不同提供了见解。最后，我们将针对大量表现出交叉抗性的变体，并增加开放类状态的构象采样，以确定结构。具有更多开放构象的结构可以作为合理设计治疗极端耐药的新型抑制剂的靶点。 HIV-1PR变异体。

DESCRIPTION (provided by applicant): HIV-1 infection is a world-wide epidemic. Although current drug therapies are effective at extending patient life, infections continue to spread, especially in non-developed countries, and the emergence of drug resistance has limited the effectiveness of many protease inhibitors. The mechanism by which the evolution of accessory mutations in HIV-1 protease (HIV-1PR) act to recover enzymatic function while imparting cross resistance to numerous inhibitors is the focus of this proposal. We are proposing a novel mechanism, which evokes protein conformational sampling, as a molecular basis to explain how secondary mutations accomplish this task. Specifically we hypothesize that mutations combine in patterns that alter HIV-1PR conformational sampling among four nominal states; namely, the closed, the semi- open, curled/tucked and wide-open such that (1) enzyme function is recovered by the secondary mutations when they combine to stabilize the semi-open conformation to a percentage observed in a native enzyme (e.g. inhibitor naive sequence) and (2) that cross resistance emerges when the mutations combine to stabilize "open-like" states such as the wide-open and curled/tucked conformations while concomitantly destabilizing the closed state population. It is through an innovative application of pulsed electron paramagnetic spectroscopy, which our lab has pioneered over the last five years that we measure conformational sampling ensembles in HIV-1PR. Within the aims of this proposal, we will make correlations among changes in HIV-1PR conformational sampling ensembles to enzymatic parameters, inhibition constants, inhibitor susceptibility, and viral fitness for numerous HIV-1PR variants. From these results, if our hypothesis is correct, we will provide a molecular level understanding of how secondary mutations elicit their effects on HIV-1PR. Additionally, these studies will also provide insights into explaining why the patterns of secondary mutation evolution against protease inhibitors are divergent in non-B subtypes. Finally, we will target numerous variants that show cross-resistance and increased conformational sampling of the open-like states for structure determination. Structures with more open-like conformations can serve as targets for the rational design of the new inhibitors for treatment of extremely resistant HIV-1PR variants.