Investigating an interface between gp120 and gp41 for HIV entry inhibition

描述（由申请人提供）：我们最近描述了一种HIV融合抑制剂PF-68742，它靶向一个涉及gp41融合肽（FP）和二硫环（DSL）区域的新位点。我们已经在gp120的C5区域发现了深刻影响HIV对PF-68742敏感性的突变。结果表明，PF-68742通过gp120和gp41之间的一个涉及FP、DSL和C5的界面与天然HIV Env结合。有趣的是，其他几种HIV进入抑制剂的抗病毒活性对涉及gp120-gp41界面和/或FP的突变表现出复杂但难以解释的敏感性。例如，VIRIP是最近描述的一种靶向FP的融合抑制剂，但HIV对VIRIP的抗性和逃逸尚未得到很好的研究，而FP的突变最近被证明深刻影响HIV对ccr5依赖性进入抑制剂（如马拉韦洛克（MVC））的敏感性。有趣的是，fp介导的对这些抑制剂的抗性或逃逸与“负”抑制有关，即在低浓度抑制剂存在下，特定HIV变体的传染性增加，随后在高浓度抑制剂下抑制。涉及gp120-gp41界面突变的不同进入抑制剂活性的功能结果的这些共性对药物开发具有影响，因此值得进一步研究。为了更好地了解PF-68742的精细特异性和作用机制，在Specific Aim 1中，我们将使用致突变分析来探索与VIRIP和MVC相比，影响HIV对PF-68742敏感性的Env结构决定因素。这些实验将阐明FP及其突变在产生多类耐药中的作用，将有助于概述FP与Env其他区域之间的功能关系，并将为下游筛选新的进入抑制剂提供信息。还将评估PF-68742和VIRIP与其他进入抑制剂的协同作用潜力。在Specific Aim 2中，我们将制备示踪标记版本的PF-68742和VIRIP，以物理探测其结合位点的特定元素在天然Env的不同激活状态下的暴露，以及用于探测具有gp120-gp41接口的相关模拟Env分子。在Specific Aim 3中，我们将筛选多种化合物文库，目的是通过结合和干扰Env中影响FP的保守亚基界面来识别抑制HIV的药物。“命中”化合物影响Env稳定性和与PF-68742、VIRIP或其他现有融合抑制剂互补或协同的可能性也将进行测试。总的来说，将获得的Env的结构-功能信息，我们将开发的工具和新的Env三聚体结合测定，以及我们将确定的新的进入抑制剂先导物，都与设计和发现HIV-1天然Env中保守的gp120-gp41界面的HIV进入抑制剂相关。

DESCRIPTION (provided by applicant): We recently described an HIV fusion inhibitor, PF-68742, that targets a novel site involving the fusion peptide (FP) and disulfide loop (DSL) region of gp41. We have since discovered mutations in the C5 region of gp120 that profoundly affect sensitivity of HIV to PF-68742. The results suggest that PF-68742 binds to native HIV Env in an interface between gp120 and gp41 involving FP, DSL and C5. Interestingly, antiviral activities of several other entry inhibitors to HIV show complex but poorly explained sensitivity to mutations involving the gp120-gp41 interface and/or FP. As examples, VIRIP is a recently described fusion inhibitor that targets FP but HIV resistance and escape to VIRIP has not been well studied, whereas mutations in FP have recently been shown to profoundly affect HIV sensitivity to CCR5-dependent entry inhibitors such as maraviroc (MVC). Interestingly, FP-mediated resistance or escape to these inhibitors has been associated with 'negative' inhibition, in which infectivity of particular variants of HIV is increased in the presence of low concentrations of inhibitor, followed by inhibition at higher concentrations. These commonalities in the functional outcome of different entry inhibitor activities involving mutations at the gp120-gp41 interface have implications for drug development and therefore warrant further investigation. To gain a better understanding of the fine specificity and mechanism of action of PF-68742, in Specific Aim 1, we will use mutagenic profiling to probe structural determinants of Env that affect HIV sensitivity to PF-68742 in comparison to VIRIP and MVC. The experiments will address the role of FP and its mutation in generating multi-class drug resistance, will help outline the functional relationship between FP and other regions of Env, and will inform downstream screens for new entry inhibitors. Potential for synergy of PF-68742 and VIRIP with other entry inhibitors will also be evaluated. In Specific Aim 2, we will prepare tracer labeled versions of PF-68742 and VIRIP to physically probe the exposure of specific elements of their binding sites in different activation states of native Env, as well as for use in probing relevant Env mimetic molecules that bear a gp120-gp41 interface. In Specific Aim 3, we will screen a diverse chemical compound library with the aim of identifying agents that inhibit HIV by binding to and perturbing the conserved subunit interfaces within Env that affect FP. Potential for 'hit' compounds to affect Env stability and complement or synergize with PF-68742, VIRIP or other existing fusion inhibitors will also be tested. Overall, the structure-function information on Env that will be gained, the tools and novel Env trimer- binding assays that we will develop, as well as the new entry inhibitor leads we will identify are relevant to the design and discovery of HIV entry inhibitors to a conserved gp120-gp41 interface in native HIV-1 Env. PUBLIC HEALTH RELEVANCE: New and better drugs are desired that can block HIV before it enters into host cells. To accelerate discovery and development of such drugs, we wish to understand the mechanisms of a recently described, first-in-class HIV entry inhibitor, and distinguish its unique features from those it shares with other key entry inhibitor drugs. We will produce valuable tools for probing its binding site, and then look for relevant new HIV drug leads.