Elucidating Molecular Mechanisms of Drug Resistance in HIV-1 Protease

描述（由申请人提供）：HIV-1感染是一种世界性的流行病。尽管目前的药物治疗在延长患者生命方面是有效的，但感染仍在继续传播，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蛋白酶（HIV-1PR）中辅助突变的进化在恢复酶功能的同时赋予对许多抑制剂的交叉抗性的机制是本提案的重点。我们提出了一种新的机制，它唤起了蛋白质构象采样，作为解释次生突变如何完成这项任务的分子基础。具体来说，我们假设突变组合在四种名义状态下改变HIV-1PR构象采样的模式；也就是说，封闭的、半开放的、卷曲/卷曲的和大开放的，这样(1)当它们结合在一起将半开放的构象稳定在天然酶中观察到的百分比时，酶的功能通过二次突变恢复（例如抑制剂初始序列）；(2)当突变结合在一起稳定“开放”的状态时，交叉抗性出现，例如大开放和卷曲/卷曲的构象，同时破坏封闭状态群体的稳定。通过脉冲电子顺磁光谱的创新应用，我们的实验室在过去的五年中率先在HIV-1PR中测量构象采样集合。在本提案的目标内，我们将在HIV-1PR构象采样集合的变化与酶参数、抑制常数、抑制剂敏感性和许多HIV-1PR变体的病毒适应度之间建立相关性。从这些结果来看，如果我们的假设是正确的，我们将从分子水平上理解继发性突变如何引发它们对HIV-1PR的影响。此外，这些研究也将为解释为什么针对蛋白酶抑制剂的二次突变进化模式在非b亚型中是不同的提供见解。最后，我们将针对显示交叉阻力和增加的开样态构象采样的许多变体进行结构确定。具有更多开放构象的结构可以作为合理设计治疗极耐药的新抑制剂的目标

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.