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

描述（由申请人提供）： 由于最近疫苗和杀菌剂试验的挫折，迫切需要减缓艾滋病毒大流行的新策略。开发一种小分子来阻断 HIV-1 复制所需的宿主过程可以提供这样的策略。与所有逆转录病毒一样，HIV-1 需要宿主蛋白来完成其生命周期，而这些细胞内宿主分子代表了尚未开发的新型抗 HIV 疗法。该应用的目标是利用功能蛋白质组学来识别宿主蛋白，这些蛋白对于宿主来说是可有可无的，但对于病毒复制至关重要。通用蛋白质组学筛选可能会鉴定出许多由 HIV 感染诱导的宿主蛋白，其中大多数都是较差的治疗候选蛋白。功能性蛋白质组学筛选可查询具有强大治疗潜力的蛋白质子集，这将大大简化对宿主靶标的搜索；宿主嘌呤组具有这种潜力。嘌呤组包括任何结合含嘌呤分子（例如ATP、NADH）的蛋白质，并且它包括热休克蛋白、脱氢酶和蛋白激酶。嘌呤体蛋白的抑制构成了许多当前疗法的基础，包括针对癌症、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体蛋白将阻止HIV-1复制。我们的研究团队设计了一种蛋白质亲和介质，可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（细胞培养物中氨基酸的稳定同位素标记）来量化THP1和Jurkat细胞中HIV-1感染诱导的嘌呤体蛋白（目标识别）；确定哪些嘌呤体蛋白对于 HIV-1 复制至关重要（目标验证）；识别选择性竞争嘌呤体结合树脂中已验证靶标的药物（药物发现）；确认这些化合物可阻断原代细胞中的 HIV 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，如果未鉴定出可行的嘌呤组靶点和/或药物，则通过更好地了解 HIV-1 与其宿主如何相互作用，病毒生物学将得到推进。拟议的研究使用创新的蛋白质组学技术 (SILAC) 和一种非常规但经过验证的方法。首先，传统的抗艾滋病毒疗法会抑制病毒编码的蛋白质，这使得它们更容易产生耐药性。通过针对 HIV-1 复制所需的宿主酶，病毒很可能必须经历更彻底的变化才能抵消宿主环境的变化。其次，传统的药物发现使用还原论方法来分别识别靶点和药物，而我们的方法结合了靶点/药物识别过程，这使得发现更加高效。该项目完成后，我们的经验和技术相结合，将使我们能够表征 HIV-1 复制所必需的宿主嘌呤结合蛋白，并鉴定出几种可抑制 HIV-1 复制所必需的宿主蛋白的先导化合物。

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.