Identifying A New Class of HIV Maturation Inhibitors

鉴定一类新的 HIV 成熟抑制剂

• 批准号: 9529507
• 负责人: Ronald I Swanstrom
• 金额: $ 23.33万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-17 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9529507
• 关键词: Active Sites; Affect; Anti-HIV Agents; Aspartic Endopeptidases; Binding; Biochemical; Biological Assay; Biological Phenomena; CCR5 gene; Capsid; Chemicals; Chimeric Proteins; Cleaved cell; Complement; Development; Drug resistance; Future; Gel; Goals; HIV; HIV Envelope Protein gp120; HIV-1; HIV-1 protease; Heterogeneity; Knowledge; Lead; Life Cycle Stages; Measures; Molecular Conformation; Monitor; Nucleosides; Pathway interactions; Peptide Fragments; Peptide Hydrolases; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Plant Roots; Poison; Polymers; Process; Protease Inhibitor; Proteins; Renin; Resistance; SP1 gene; Site; Specificity; Structure; System; Testing; Therapeutic Index; Thinking; Viral; Viral Proteins; Virion; Virus; Virus Replication; Work; anti-cancer; base; clinical development; complex biological systems; drug discovery; experience; fitness; gag Gene Products; genetic approach; high throughput screening; inhibitor/antagonist; internal control; non-nucleoside reverse transcriptase inhibitors; novel; novel therapeutics; protein protein interaction; screening; secondary analysis; small molecule inhibitor; tool; virtual

Abstract Effect anti-HIV drugs have focused on a limited number of targets. While these drugs are currently highly efficacious there is a need to continue to develop new targets for two reasons. First, it is possible that over the course of treating infected people for the decades to come drug resistance could become a problem; thus developing new targets and inhibitors now provides the theoretical underpinning for the development of new drugs in the future. Second, inhibitors represent chemical probes that can complement genetic approaches in terms of studying biological phenomena. Thus inhibitors that are not drugs still have intrinsic value as tools in understanding complex biological systems. We have previously shown that the MA/CA protease cleavage site in the HIV-1 Gag protein must be cleaved to near completion to allow an infectious virus particle to form. Even 10% under-cleavage of this site poisons the assembly process, likely because some of the uncleaved MA/CA fusion protein participates in capsid formation and poisons this highly regulated process. Bevirimat formed a conceptual basis for thinking about inhibitors of specific cleavage sites, in this case CA/SP1. However, among all of the cleavage sites in Gag the MA/CA cleavage site is by far the most sensitive to under-processing to affect infectivity. For this reason we undertook to develop a biochemical assay that could be used in a high throughput screen for small molecule inhibitors. That screen (775,000 compounds screened by SRI) is now completed with the most active compounds having an IC50 starting at 1 uM. These compounds have already been shown not to be HIV-1 protease inhibitors. In this application we propose four aims. First, we will test the most active compounds in a secondary screen using our two-substrate gel-based assay that includes both the target MA/CA substrate and as an internal control a substrate that can be cleaved by the HIV-1 protease but which should not be a target of the inhibitor. Second, we will measure EC50s for viral replication using a novel assay that will greatly enhance our sensitivity to detect specific inhibition. Third, we will measure EC50s for a panel of transmitted/founder viruses and viruses from different clades to provide an initial assessment of the effect of sequence variability on sensitivity. And fourth, for those compounds that have a sufficient therapeutic index we will start to select for resistance to validate the MA/CA target. With this effort we will identify the most promising hits in this new screen that can then be used in the future as the basis for structural studies and SAR.

抽象的 抗艾滋病毒药物的作用集中在有限的几个靶点上。虽然这些药物目前高度 由于两个原因，需要继续制定新的目标。首先，有可能超过 未来几十年治疗感染者的过程中，耐药性可能会成为一个问题；因此 开发新的靶点和抑制剂现在为开发新的药物提供了理论基础 未来的药物。其次，抑制剂代表化学探针，可以补充遗传方法 研究生物现象的术语。因此，非药物抑制剂仍然具有作为治疗疾病的工具的内在价值。 了解复杂的生物系统。我们之前已经表明 MA/CA 蛋白酶切割位点 HIV-1 Gag 蛋白中的部分必须被切割至接近完全才能形成感染性病毒颗粒。甚至 该位点 10% 的裂解不足会毒害组装过程，可能是因为一些未裂解的 MA/CA 融合蛋白参与衣壳形成并毒害这一高度调控的过程。贝维里马特成立了 这是考虑特定切割位点（在本例中为 CA/SP1）抑制剂的概念基础。然而，其中 Gag 中的所有切割位点中，MA/CA 切割位点是迄今为止对处理不足最敏感的 影响传染性。为此，我们着手开发一种可用于高浓度的生化检测方法。 小分子抑制剂的通量筛选。该筛选（SRI 筛选了 775,000 种化合物）现已 使用IC50从1 uM开始的最活跃的化合物完成。这些化合物已经 已被证明不是 HIV-1 蛋白酶抑制剂。在此应用中，我们提出了四个目标。首先，我们将测试 使用我们的基于双底物凝胶的测定法进行二次筛选中最活跃的化合物，其中包括 目标 MA/CA 底物，作为内部对照，底物可被 HIV-1 蛋白酶切割，但 这不应该是抑制剂的目标。其次，我们将使用一种新颖的方法来测量病毒复制的 EC50 这将大大提高我们检测特异性抑制的灵敏度。第三，我们将测量 EC50 传播/创始病毒和来自不同进化枝的病毒小组，以提供对病毒的初步评估 序列变异性对灵敏度的影响。第四，对于那些具有足够治疗作用的化合物 我们将开始选择阻力位来验证 MA/CA 目标。通过这一努力，我们将确定最 这个新屏幕中的热门产品可以在未来用作结构研究的基础 特别行政区。