CVN-12p1 Chimeras and Combinations for AIDS Microbiocides

描述(申请人提供)：在全球范围内，艾滋病毒和艾滋病最主要的传播途径是异性性交。杀微生物剂是一种可用于阴道和直肠配方的化合物，越来越多地被视为阻止传播的紧迫目标。氰病毒蛋白-N(CV-N)是一种高度稳定的蛋白，可与HIV-1Env gp120结合，对抗病毒进入宿主细胞和随后的感染。CV-N可大规模重组生产，已被发现在动物研究中有效阻止HIV-1传播，目前正处于临床前开发阶段，作为一种杀微生物剂。尽管如此，仍然存在潜在的限制，包括根据在体内测量的效力所需的CV-N生产量、对单一作用模式的依赖以及对CV-N具有耐药性的病毒株。通过将该蛋白与第二种抑制剂结合到同一分子靶标gp120中的不同相互作用部位，并协同其与CV-N的拮抗活性，可以克服CV-N的局限性。最近，我们发现一种名为12p1的多肽可以作为gp120与宿主细胞受体相互作用的变构双重拮抗剂，并能提高CV-N在混合物中的抗病毒活性。我们已经确定了一种化学修饰策略，该策略可以产生与gp120有显著高亲和力的12p1形式。此外，我们有领先的数据表明，12p1和CV-N的重组嵌合体比单独的CVN具有更强的拮抗活性。这项建议的R21部分是研究进化的基于12p1的分子作为增强CV-N作为杀菌剂的能力的可能手段的有用性。R33阶段将开发有前景的铅组合和嵌合体，包括基于结构作用机制的先进试剂设计；确定铅嵌合体和组合的体外和体内效力，并审查与杀微生物剂领域其他制剂的协同作用；以及为本工作中衍生的蛋白质和多肽制剂开发生产方法。 我们将在R21项目中优先考虑两个具体目标。[1]生产具有不同连接子的重组CVN-12p1嵌合体，并测定候选嵌合体相对于单独的CV-N和12p1组分的有效性。这一目标的工作将确定具有优化的HIV-1环境gp120拮抗作用和潜在的重组艾滋病杀微生物剂候选的CVN-12p1嵌合体。[2]产生并筛选用于与CVN非共价结合的亲和力增强的共轭12p1变体，并鉴定优化的混合物候选。这一目标的工作将提供将重组CV-N与合成的12P1结合物相结合的替代杀微生物剂候选。 在R21阶段结束时的主要里程碑将是鉴定12P1/CVN组合，包括嵌合体和混合物，它们的拮抗活性将比CV-N单独增强，因此将被推荐作为本身的杀微生物剂开发。CVN-12p1嵌合体和非共价组合将引入一种具有新作用机制的杀微生物剂，基本上克服了单独使用CV-N的效力和抗药性限制，从而导致改进的杀微生物剂候选方案，以对抗全球艾滋病传播。

DESCRIPTION (provided by applicant): The most dominant means of transmission of HIV and spread of AIDS worldwide is by heterosexual intercourse. Microbicides, compounds that could be used in vaginal and rectal formulations, are increasingly seen as an urgent goal to stop transmission. The protein cyanovirin-N (CV-N) is a highly stable protein that binds to HIV-1 Env gp120 and antagonizes viral entry into host cells and consequent infection. CV-N can be produced recombinantly at large scale, has been found to be effective in blocking HIV-1 transmission in animal studies and is currently in pre-clinical development as a microbicide. Nonetheless, there are potential limitations, including amount of CV-N production required based on measured in vivo efficacy, reliance on a single mode of action and CV-N resistant strains of virus. Limitations of CV-N could be overcome by combining this protein with a second inhibitory agent that binds to a different interaction site in the same molecular target, gp120, and synergizes its antagonist activity with that of CV-N. Recently, we have found that a peptide denoted 12p1 functions as an allosteric dual antagonist of gp120 interactions with host cell receptors and can improve the antiviral activity of CV-N in combination mixtures. We have identified a chemical modification strategy that yields strikingly higher affinity forms of 12p1 for gp120. Further, we have lead data demonstrating that a recombinant chimera of 12p1 and CV-N has enhanced antagonist activity vs CVN alone. The R21 part of this proposal is to examine the usefulness of evolving 12p1-based molecules as possible means to enhance the capability of CV-N as a microbicidal agent. The R33 phase will be to develop promising lead combinations and chimeras, including advanced agent design based on structural mechanism of action; to define in vitro and in vivo efficacy of lead chimeras and combinations and to examine synergies with other agents in the microbicide field; and to develop production approaches for the protein and peptide agents derived in this work. We will prioritize two specific aims in the R21 project. [1] Produce recombinant CVN-12p1 chimeras with varying linkers and determine efficacy of chimera candidates versus that of the separate CV-N and 12p1 components. Work on this aim will identify CVN-12p1 chimeras with optimized HIV-1 Env gp120 antagonism and potential as recombinant AIDS microbicide candidates. [2] Generate and screen affinity-enhanced conjugate 12p1 variants for non-covalent combination with CVN and identify optimized mixture candidates. Work in this aim will provide alternative microbicide candidates that combine recombinant CV-N with synthetic 12p1 conjugates. The major milestone at the end of the R21 phase will be identification of 12p1/CVN combinations, both chimeras and mixtures, which will have antagonist activities that are enhanced vs CV-N alone and which hence will be recommended for development as microbicides in their own right. The CVN-12p1 chimeras and noncovalent combinations will introduce a microbicide with a novel mechanism of action, substantially overcome potency and resistance limitations of CV-N alone and hence lead to improved microbicide candidates to combat global AIDS transmission.