Basic and Comparative Studies of CCR5 Inhibition to Prevent HIV Transmission

描述(申请人提供)：艾滋病毒大流行的增长，主要通过性传播，迫切需要制定战略，以限制其爆炸性传播。显然，目前还没有一种单一的战略可以实现这一点。因此，至关重要的是继续在多个层面和多个学科开展研究，以制定可能有助于减少艾滋病毒传播的预防战略。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，这些项目将提供新的信息，这些信息对于制定一般的杀微生物剂战略、针对CCR5的战略以及更好地了解人类和非人类灵长类女性生殖道至关重要。我们一直在开发RANTES类似物，以亚纳摩尔浓度阻断OCR并抑制HIV复制。我们的第一个线索，PSC-RANTES已经保护了用SIV 162P3挑战的10只恒河猴中的全部10只，从而提供了迄今为止报告的任何毒剂中对SIV最有效的保护。这一战略的局限性包括对不同RANTES类似物阻止艾滋病毒获得CCR5的基本机制的不完全了解，它们可能导致炎症的潜在激动剂活性，保护的有限持久性，合成的成本，以及最后对人类和恒河猴女性生殖道之间的异同的不完全了解，这可能限制在一个中的发现推广到另一个中的应用。在这里，我们建议通过三个项目来解决这些问题：项目1：定义新型CCRs-靶向杀菌剂候选的抑制机制-由于我们已经开发了几类对CCR5定位和信号转导有谨慎不同影响的RANTES类似物，这些研究旨在探索这些类似物活性背后的细胞和分子相互作用。项目2：人类和猕猴的妇科比较研究--这将侧重于确定人类和非人类灵长类动物的宫颈阴道上皮之间的异同，以确定在多大程度上可以将这些动物的发现转化为人类的活动预测。因此，这项工作的结果对于开发RANTES类似物以及所有其他局部预防艾滋病毒传播的战略都是至关重要的。项目3：开发RANTES类似物作为预防艾滋病毒传播的局部策略：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，将检查艾滋病毒抑制剂组合和新型水凝胶配方的协同活性，以增强保护的持久性，并将开发大规模合成完全重组剂的GMP方法，从而能够负担得起这一策略的应用。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。 项目1：确定新型CCRs靶向杀菌剂候选的抑制机制，Mosier，D. 项目1说明(申请人提供)：项目1的目标是确定三个新的趋化因子类似物的作用机制，这些类似物具有有效阻止艾滋病毒-1通过CCR5进入的活性。这些完全重组的分子表现出三种不同的活性：(I组)CCR5被阻断而不具有信号活性或受体内化；(II组)CCR5快速内化并具有信号活性；(III)适度内化和阻断CCR5而不具有信号活性。这些新分子在安全性和生产成本方面比目前的抑制剂(如PSC-RANTES)具有显著的潜在优势，但这些候选杀菌剂的进一步改进需要更详细的作用机理知识。我们将确定II族分子的细胞内隔离途径，并与III族分子进行比较。我们将检验一些假说，以解释在没有细胞内受体隔离(组I)或适度内化(组III)的情况下持续的抗病毒活性，包括CCR5二聚体形成的变化，膜上受体定位的改变，变构效应，以及独立于G蛋白连接的信号的受体内化。我们还将研究CCR5、CCL5和CCL3L1基因多态对CCR5蛋白质合成和周转率的影响。这些研究旨在调查来自正常人类捐赠者的初级靶细胞对每种新抑制剂的敏感性的变异性。我们还将使用一组突变的CCR5分子来检查活性的结构相关性。我们将利用这些关于机制和靶细胞可变性的信息来开发具有更好活性特征的新分子，并将与该计划中的其他项目进行协调实验，以将我们在基于细胞的模型中的发现与当前和新的CCR5抑制剂在组织外植体和整个动物模型中的效果联系起来。这一方法将产生更好和更安全的CCR5抑制剂，可按适合于阻止艾滋病毒/艾滋病在受影响最严重地区传播的规模生产。

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.