Rescuing the anti-HIV activity of APOBEC3G with small-molecule chaperons

描述(申请人提供)：在这项提案中，我们计划评估基于人类细胞的内在防御机制开发抗病毒化疗策略的潜力：用胞苷脱氨酶APOBEC3G(A3G)诱变单链DNA。人类免疫缺陷病毒1(HIV-1)通过表达病毒粒子感染性因子(Vif)来对抗这种活性，病毒粒子感染性因子下调A3G的活性和稳定性。Vif在病毒增殖中的重要作用和A3G的高度调控功能使其相互作用成为无耐药性干预的一个有吸引力的靶点。此外，通过3G诱导的突变在原位产生显性阴性HIV-1突变的可能性为灭活潜伏感染细胞中的HIV提供了一个独特的机会，通常无法获得化疗药物。这项拟议的研究基于这样的假设，即能够与Vif竞争A3G的小分子将同时保护细胞因子免受Vif的拮抗，并降低病毒因子的稳定性。这项提案将通过利用遗传选择的独特力量来产生稀有的解决方案，来解决确定蛋白质-蛋白质相互作用的从头开始抑制物的挑战。因此，我们相信，这一假说的实验验证提供了一个独特的机会，可以通过潜伏感染细胞中的自然防御机制来遏制艾滋病毒感染的进展，而不会有进化耐药表型的危险。我们计划在发展这一方法时实现以下具体目标： 具体目标1：我们将开发和实施一种遗传选择方案，以发现Vif-A3G相互作用的有效和选择性拮抗剂。 具体目标2：我们将通过一系列生化和细胞分析来确认和表征所选主干环肽的Vif降解和/或A3G保护活性。 具体目标3：我们将确定对所选序列的活性重要的功能基序，并开发它们的细胞通透性衍生物。 这项研究的预期结果将是一系列特定和有效的细胞渗透剂，能够保护A3G免受VIF下调的影响。我们的长期目标是开发一种新型抗艾滋病毒药物的原型，以解决与当前策略相关的三个现有问题：毒性、耐药性和潜伏期。为了保护生物体免受病毒感染，人类细胞开发了一种复杂的防御机制，涉及对病毒基因的修饰，这对病毒复制是致命的。不幸的是，人类免疫缺陷病毒(HIV)能够抵消这种内在保护系统，使其引起的疾病--获得性免疫缺陷综合症(AIDS)--成为一个全球性的健康问题，目前还看不到治愈或预防的迹象。我们建议开发一种新的抗HIV治疗策略，该策略将挽救自然的抗病毒机制，而不会产生抗药性病毒的危险。

DESCRIPTION (provided by applicant): In this proposal, we plan to evaluate the potential for developing an antiviral chemotherapeutic strategy based on the intrinsic defense mechanism of human cells: mutagenesis of single-stranded DNA by cytidine deaminase APOBEC3G (A3G). Human immunodeficiency virus 1 (HIV-1) counters this activity by expressing virion infectivity factor (Vif) that downregulates both the activity and stability of A3G. The essential role of Vif in viral proliferation and highly regulated function of A3G make their interaction an attractive target for the resistance-free intervention. Furthermore, a possibility of generating dominant negative HIV-1 mutants in situ through A3G-induced mutagenesis presents a unique opportunity to inactivate HIV in latently infected cells, normally inaccessible to chemotherapeutics. The proposed research is based on the hypothesis that small molecules capable of competing with the Vif for A3G will simultaneously protect the cellular factor from the antagonism displayed by Vif and reduce stability of the viral factor. The challenge of identifying de novo inhibitors of protein-protein interaction will be addressed in this proposal by exploiting the unique power of genetic selection to yield rare solutions. We believe, therefore, that experimental validation of this hypothesis presents a unique opportunity for curbing the progression of HIV infections through a natural defense mechanism in latently infected cells without the danger of evolving resistant phenotypes. We plan to accomplish the following Specific Aims in developing this approach: Specific Aim 1: We will develop and implement a genetic selection scheme for the discovery of potent and selective antagonists of Vif-A3G interaction. Specific Aim 2: We will confirm and characterize Vif-degrading and/or A3G-protecting activities of the selected backbone cyclic peptides using a series of biochemical and cellular assays. Specific Aim 3: We will identify functional motifs important for the activity of the selected sequences and develop their cell-permeable derivatives. The expected outcome of this research will be a series of specific and potent cell-permeable agents capable of protecting A3G from the downregulation by Vif. Our long-term objective is to advance a prototype of a new class of anti-HIV drugs that can address the three existing problems associated with the current strategies: toxicity, resistance and latency. To protect organism against viral infections, human cells have developed a sophisticated defense mechanism involving modifications of viral genes, lethal to virus replication. Unfortunately, human immunodeficiency virus (HIV) is able to counteract this intrinsic protective system making the disease that it causes, Acquired Immunodeficiency Syndrome (AIDS), a global health problem, with no cure or prevention in sight. We propose to develop a novel anti-HIV therapeutic strategy that will rescue the natural antiviral mechanism without the danger of producing drug-resistant viruses.