CVN-12p1 Chimeras and Combinations for AIDS Microbiocides

描述（由申请人提供）：在世界范围内，艾滋病毒传播和艾滋病传播最主要的途径是异性性交。杀微生物剂，可用于阴道和直肠配方的化合物，越来越被视为阻止传播的紧迫目标。cyanovirin-N （CV-N）蛋白是一种高度稳定的蛋白，可与HIV-1 Env gp120结合，并对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，在动物研究中已被发现在阻断HIV-1传播方面有效，目前正作为杀微生物剂进行临床前开发。尽管如此，仍存在潜在的局限性，包括基于体内有效性测量所需的CV-N产量，对单一作用模式的依赖以及对CV-N具有抗性的病毒株。通过将这种蛋白与第二种抑制剂结合，可以克服CV-N的局限性，第二种抑制剂结合到相同分子靶标gp120的不同相互作用位点，并与CV-N的拮抗剂活性协同。最近，我们发现一种名为12p1的肽可作为gp120与宿主细胞受体相互作用的变构双拮抗剂，并可在联合混合物中提高CV-N的抗病毒活性。我们已经确定了一种化学修饰策略，可以产生对gp120具有更高亲和力的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具有增强的拮抗剂活性。本提案的R21部分是研究进化的12p1基分子作为增强CV-N作为杀微生物剂能力的可能手段的有用性。R33阶段将是开发有前景的先导组合和嵌合体，包括基于作用结构机制的高级药物设计；确定铅嵌合体及其组合在体外和体内的功效，并检查与杀菌剂领域其他药剂的协同作用；并开发本工作衍生的蛋白质和肽制剂的生产方法。

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.