Basic and Comparative Studies of CCR5 Inhibition to Prevent HIV Transmission

描述（由申请人提供）：主要通过性行为传播的艾滋病毒流行病的增长，迫切需要制定限制其爆炸性传播的战略。很明显，目前还没有一个单一的战略能够做到这一点。因此，至关重要的是继续在多个层次和学科进行研究，以制定可能有用的预防战略，减少艾滋病毒的传播。我们组建了一个优秀的团队，他们提出了三个相互关联的项目，这些项目将为总体杀菌剂策略的开发、针对CCR5的策略以及更好地了解人类和非人类灵长类动物女性生殖道提供重要的新信息。我们一直在开发阻断OCRS并在亚纳摩尔浓度下抑制HIV复制的RANTES类似物。我们的第一个线索，PSC-RANTES已经保护了10只感染SHIV 162P3的恒河猴中的所有10只，因此提供了迄今为止报道的任何药剂中最有效的针对SHIV的保护。该策略的局限性包括对不同RANTES类似物阻断HIV进入CCR5的基本机制的不完全理解，它们可能导致炎症的潜在激动剂活性，有限的保护持久性，合成成本以及最终对人类和恒河猴女性生殖道之间的相似性和差异的不完全理解，这可能限制了一种发现的推广应用于另一种。在这里，我们建议通过三个项目来解决这些问题：项目1：确定新型ccrs靶向杀菌剂候选物的抑制机制-由于我们已经开发了几种对CCR5定位和信号传导有不同影响的RANTES类似物，这些研究旨在探索这些类似物活性基础上的细胞和分子相互作用。项目2：人类和恒河猴的比较妇科研究-这将侧重于定义人类和非人类灵长类动物的宫颈阴道上皮的异同，以确定这些动物的发现可以在多大程度上转化为人类活动的预测。因此，这项工作的结果对于RANTES类似物的开发以及所有其他局部预防艾滋病毒传播的策略都至关重要。项目3：开发RANTES类似物作为局部预防艾滋病毒传播的策略：将探索不同类似物的激动剂活性，以确定潜在炎症效应的抑制途径，将研究艾滋病毒抑制剂组合的协同活性和新型水凝胶配方，以增强保护的持久性，并将开发完全重组药物的大规模GMP合成方法，使该策略的应用能够负担得起。经验丰富的行政核心为这些项目提供基础设施支持，并为分析其结果提供统计专门知识。

DESCRIPTION (provided by applicant): Growth of the HIV pandemic that is largely transmitted through sex, gives a compelling need to develop strategies to limit its explosive spread. It is clear that there is not yet a single strategy that will accomplish this. Thus it is vital to continue research at multiple levels and disciplines to develop potentially useful prevention strategies that may provide attenuation of HIV transmission. We have assembled an outstanding team who propose three interlocking projects that will provide new information critical to the development of microbicide strategies in general, to strategies targeting CCR5 and to developing a better understanding of the human and non-human primate female genital tract. We have been developing RANTES analogues that block OCRS and inhibit HIV replication at subnanomolar concentrations. Our first lead, PSC-RANTES has protected all 10 of 10 rhesus macaques challenged with SHIV 162P3 thus providing the most potent protection against SHIV of any agent reported to date. Limitations to this strategy include an incomplete understanding of the fundamental mechanisms whereby different RANTES analogues block HIV access to CCR5, their potential agonist activity that may result in inflammation, a limited durability of protection, the costs of synthesis and finally an incomplete understanding of the similarities and differences between the human and rhesus female genital tracts that may limit generalization of findings in the one to application in the other. Here, we propose to address each of these issues, with three projects: Project 1: Defining inhibitory Mechanisms of Novel CCRS-targeted Microbicide Candidates - As we have developed several classes of RANTES analogues with discreetly different effects on CCR5 localization and signaling, these studies are designed to explore the cellular and molecular interactions that underlie the activities of these analogues. Project 2: Comparative gynecologic studies in humans and rhesus macaques - This will focus on defining the similarities and differences between the cervicovaginal epithelium of humans and non-human primates to establish the degree to which findings in these animals can be transposed to projections of activity in humans. Thus, the results of this work will be critical both to the development of RANTES analogues as well as to all other strategies for topical prevention of HIV transmission. Project 3: Developing RANTES analogues as topical strategies to prevent HIV transmission: will explore agonist activities of different analogues to identify inhibitory pathways of potential inflammatory effects, will examine synergistic activities of HIV inhibitor combinations and novel hydrogel formulations to enhance the durability of protection and will develop methods for large scale GMP synthesis of fully recombinant agents that will permit affordable application of this strategy. An experienced Administrative Core provides infrastructural support for these projects as well as statistical expertise for analyses of their results. PROJECT 1: Defining Inhibitory Mechanisms of Novel CCRS-targeted Microbicide Candidates, Mosier, D. PROJECT 1 DESCRIPTION (provided by applicant): The goal of Project 1 is to define the mechanism of action of three new chemokine analogues with potent activity in blocking HIV-1 entry via CCR5. These fully recombinant molecules display three distinct activity profiles: (group I) CCR5 blockade without signaling activity or receptor internalization; (group II), CCR5 rapid internalization with signaling activity; and (group III), moderate CCR5 internalization and blockade without signaling activity. These new molecules have significant potential advantages in terms of safety and cost of production over current inhibitors such as PSC-RANTES, but further improvements on these candidate microbicides requires more detailed knowledge of their mechanisms of action. We will define the route to intracellular sequestration for group II molecules, and compare with that of the group III molecules. We will examine a number of hypotheses to explain prolonged antiviral activity in the absence of intracellular receptor sequestration (group I) or with moderate internalization (group III), including changes in CCR5 dimer formation, altered receptor localization in the membrane, allosteric effects, and receptor internalization independent of G-protein-linked signaling. We will also examine the impact of CCR5, CCL5, and CCL3L1 genetic polymorphisms on CCR5 protein synthesis and turnover rate. These studies are designed to investigate variability in the susceptibility of primary target cells from normal human donors to each of the new inhibitors. We will also use a panel of mutant CCR5 molecules to examine structural correlates of activity. We will use this information on mechanism and target cell variability to develop new molecules with even better activity profiles, and we will perform coordinated experiments with other projects in this Program to relate our findings in cell-based models to the effects of current and new CCR5 inhibitors in tissue explant and whole animal models. This approach will generate better and safer CCR5 inhibitors that can be produced on a scale suitable for stopping the spread of HIV/AIDS in the most-impacted areas.