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大流行。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。像所有的逆转录病毒一样，HIV-1需要宿主蛋白来完成它的生命周期，而这些细胞内的宿主分子代表着一个未开发的新型抗艾滋病毒治疗药物池。这项应用的目标是使用功能蛋白质组学来识别对宿主来说是必不可少的、但对病毒复制至关重要的宿主蛋白。通用蛋白质组筛查可能会识别由艾滋病毒感染诱导的大量宿主蛋白，其中大多数将是糟糕的候选治疗方案。功能蛋白质组学筛选，查询具有强大治疗潜力的蛋白质子集，将极大地简化寻找宿主靶标的工作；宿主嘌呤核糖体具有这种潜力。嘌呤小体包括与含嘌呤分子(如ATP、NADH)结合的任何蛋白质，它包括热休克蛋白、脱氢酶和蛋白激酶。抑制嘌呤蛋白形成了目前许多治疗方法的基础，包括针对癌症、高血压和细菌感染的治疗。这一应用的中心假设是，抑制由艾滋病毒感染诱导或调节的嘌呤蛋白，将阻止艾滋病毒-1的复制。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大的步骤进行：使用SILAC(细胞培养中氨基酸的稳定同位素标记)对艾滋病毒-1感染THP1和Jurkat细胞诱导的嘌呤蛋白质进行量化(靶标鉴定)；确定哪些嘌呤蛋白质是艾滋病毒-1复制所必需的(靶标确认)；确定选择性竞争嘌呤糖体结合树脂有效靶标的药物(药物发现)；确认这些化合物阻止艾滋病毒在原代细胞中的复制(药物验证)。在这一过程中，我们还将确定与HIV-1相互作用的宿主蛋白，如果没有确定可行的嘌呤体靶向和/或药物，将通过更好地了解HIV-1与宿主相互作用的方式来推动病毒生物学的发展。这项拟议的研究使用了创新的蛋白质组学技术(SILAC)和一种非传统的、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，这使它们更容易产生耐药性。通过针对HIV-1复制所需的宿主酶，很可能病毒将不得不经历更彻底的变化，以抵消宿主环境的变化。第二，传统的药物发现使用简化论的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，这使得发现更有效率。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对HIV-1复制至关重要的宿主嘌呤结合蛋白，并识别将抑制HIV-1复制所需的宿主蛋白的几种先导化合物。

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.