A Method to Stop HIV Replication:Inhibition of Human Purine Utilizing Proteins

描述（由申请人提供）： 由于最近在疫苗和杀微生物剂试验方面的挫折，迫切需要新的战略来减缓艾滋病毒的流行。开发一种能够阻断HIV-1复制所需的宿主过程的小分子可以提供这样一种策略。像所有逆转录病毒一样，HIV-1需要宿主蛋白质来完成其生命周期，这些细胞内宿主分子代表了一种未开发的新型抗HIV治疗剂。本申请的目的是使用功能蛋白质组学来鉴定对宿主有害但对病毒复制必不可少的宿主蛋白质。一个通用的蛋白质组学筛选可能会确定许多宿主蛋白诱导的艾滋病毒感染，其中大多数将是穷人的治疗候选人。一个功能蛋白质组学的屏幕，查询一个子集的蛋白质具有强大的治疗潜力，将大大简化搜索主机的目标，主机嘌呤组具有这种潜力。嘌呤组包括结合含嘌呤分子（例如ATP、NADH）的任何蛋白质，并且其包括热休克蛋白、脱氢酶和蛋白激酶。嘌呤组蛋白的抑制形成了许多当前疗法的基础，包括靶向癌症、高血压和细菌感染的疗法。本申请的中心假设指出，抑制由HIV感染诱导或调节的嘌呤组蛋白将阻断HIV-1复制。我们的研究团队设计了一种蛋白质亲和介质，通过其嘌呤结合口袋捕获整个嘌呤组。拟议的研究将分四个主要步骤进行：使用SILAC（在细胞培养物中用氨基酸进行稳定同位素标记）来定量由HIV-1感染THP 1和Jurkat细胞诱导的嘌呤组蛋白（靶点鉴定）;确定哪些嘌呤组蛋白对HIV-1复制至关重要（靶点验证）;从嘌呤组结合树脂中鉴定选择性竞争经验证靶点的药物（药物发现）;确认这些化合物阻断HIV在原代细胞中的复制（药物验证）。在此过程中，我们还将鉴定与HIV-1相互作用的宿主蛋白，如果没有鉴定出可行的嘌呤组靶点和/或药物，将通过更好地了解HIV-1如何与宿主相互作用来推进病毒生物学。这项研究使用了创新的蛋白质组学技术（SILAC）和一种非传统但经过验证的方法。首先，传统的抗艾滋病毒疗法抑制病毒编码的蛋白质，这使得它们更容易产生耐药性。通过靶向HIV-1复制所需的宿主酶，病毒可能必须经历更彻底的变化，以抵消宿主环境的变化。其次，传统的药物发现使用还原论的方法来分别识别靶标和药物，而我们的方法结合了靶标/药物识别过程，这使得发现更有效。在这个项目完成后，我们的经验和技术的结合将使我们能够表征宿主嘌呤结合蛋白，这是HIV-1复制所必需的，并确定几个先导化合物，将抑制HIV-1复制所必需的宿主蛋白。

DESCRIPTION (provided by applicant): Owing to the recent setbacks in vaccine and microbicide trails, novel strategies to slow the HIV-pandemic are desperately needed. Development of a small molecule that blocked a host process required for HIV-1 replication could provide such a strategy. Like all retroviruses, HIV-1 requires host proteins to complete its life cycle, and these intracellular host molecules represent an undeveloped pool of novel anti-HIV therapeutics. The goal of this application is to use functional proteomics to identify host proteins that are dispensable to the host but essential for viral replication. A generic proteomic screen would likely identify numerous host proteins induced by HIV-infection, most of which would be poor therapeutic candidates. A functional proteomics screen, which queries a subset of proteins with strong therapeutic potential, would greatly simplify the search for host targets; the host purinome has this potential. The purinome comprises any protein that binds purine- containing molecules (e.g. ATP, NADH), and it includes heat shock proteins, dehydrogenases, and protein kinases. Inhibition of purinome proteins forms the basis of many current therapies, including those targeting cancer, hypertension, and bacterial infections. The central hypothesis of this application states that inhibition of purinome proteins, which are induced or regulated by HIV-infection, will block HIV-1 replication. Our research team has designed a protein affinity media that captures the entire purinome through its purine-binding pocket. The proposed research will proceed in four broad steps: use SILAC (stable isotope labeling with amino acids in cell culture) to quantify purinome proteins induced by HIV-1 infection of THP1 and Jurkat cells (target identification); determine which purinome proteins are essential for HIV-1 replication (target validation); identify drugs that selectively compete validated targets from the purinome-binding resin (drug discovery); confirm that these compounds block HIV-replication in primary cells (drug validation). During this process, we will also identify host proteins that interact with HIV-1, and in the event that a viable purinome target and/or drug is not identified, viral biology will be advanced by a greater understanding of how HIV-1 interacts with its host. The proposed research uses innovative proteomics technology (SILAC) and an unconventional, yet validated, approach. First, conventional anti-HIV therapeutics inhibit virally-encoded proteins, which makes them more susceptible to the development of resistance. By targeting a host enzyme that is required for HIV-1 replication, it is likely that the virus will have to undergo more radical changes to offset the changes in the host milieu. Second, conventional drug discovery uses a reductionist approach to separately identify a target and a drug, while our approach combines the target/drug identification process, which makes discovery more efficient. At the completion of this project, our combination of experience and technology will enable us to characterize host purine-binding proteins that are essential for HIV-1 replication and identify several lead compounds that will inhibit host proteins necessary for HIV-1 replication.