CVN-12p1 Chimeras and Combinations for AIDS Microbiocides

描述（由申请人提供）：艾滋病毒传播和艾滋病传播的最主要途径是通过异性性交。杀微生物剂，可用于阴道和直肠制剂的化合物，越来越被视为阻止传播的紧迫目标。蛋白氰病毒蛋白-N（CV-N）是一种高度稳定的蛋白，其结合HIV-1 Env gp120并拮抗病毒进入宿主细胞和随后的感染。CV-N可以大规模重组生产，在动物研究中发现它能有效阻断HIV-1传播，目前正处于临床前开发阶段。然而，存在潜在的限制，包括基于测量的体内功效所需的CV-N产生量、对单一作用模式的依赖和CV-N抗性病毒株。CV-N的局限性可以通过将这种蛋白质与第二种抑制剂结合来克服，所述第二种抑制剂结合到相同分子靶标gp120中的不同相互作用位点，并使其拮抗剂活性与CV-N的拮抗剂活性协同。最近，我们已经发现，一个肽表示为12p1的功能作为gp120与宿主细胞受体相互作用的变构双重拮抗剂，并可以提高CV-N的抗病毒活性的组合混合物。我们已经确定了一种化学修饰策略，产生了显着更高的亲和力形式的12p1的gp120。此外，我们有领先的数据表明，12p1和CV-N的重组嵌合体与单独的CVN相比具有增强的拮抗剂活性。该提案的R21部分是研究发展基于12p1的分子作为增强CV-N作为杀微生物剂的能力的可能手段的有用性。R33阶段将开发有前景的先导化合物和嵌合体，包括基于结构作用机制的先进药剂设计;确定先导化合物和嵌合体的体外和体内功效，并检查与杀微生物剂领域其他药剂的协同作用;并开发本工作中衍生的蛋白质和肽药剂的生产方法。 我们将优先考虑R21项目中的两个具体目标。[1]产生具有不同接头的重组CVN-12p1嵌合体，并确定嵌合体候选物相对于单独CV-N和12p1组分的功效。这一目标的工作将鉴定具有优化的HIV-1 Env gp120拮抗作用和作为重组AIDS杀微生物剂候选物的潜力的CVN-12p1嵌合体。[2]生成并筛选亲和力增强的缀合物12p1变体，用于与CVN非共价结合，并鉴定优化的混合物候选物。这一目标的工作将提供将联合收割机重组CV-N与合成12 p1缀合物结合的替代杀微生物剂候选物。 R21阶段结束时的主要里程碑将是鉴定12p1/CVN组合，嵌合体和混合物，其拮抗剂活性相对于单独的CV-N增强，因此将推荐其本身作为杀微生物剂开发。CVN-12p1嵌合体和非共价组合将引入具有新作用机制的杀微生物剂，基本上克服单独CV-N的效力和抗性限制，从而导致改进的杀微生物剂候选物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.