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HIV药物的效应集中在有限数量的靶标上。这些药物目前高度高 有效的是，有两个原因需要继续开发新目标。首先，有可能 几十年来，治疗受感染的人的耐药性可能成为一个问题。因此 现在，开发新的目标和抑制剂为开发新的基础提供了理论的基础 将来的毒品。其次，抑制剂代表可以补充遗传方法的化学探针 研究生物学现象的条款。因此，非药物的抑制剂仍然具有内在价值作为工具 了解复杂的生物系统。我们以前已经表明MA/CA蛋白酶裂解位点 在HIV-1中，必须将蛋白裂解至接近完成，以使传染性病毒颗粒形成。甚至 该站点的分离不足10％，可能是因为一些未泄漏的MA/CA 融合蛋白参与衣壳形成，毒药这一高度调节的过程。 bevirimat形成a 考虑特定切割部位的抑制剂的概念基础，在这种情况下为CA/SP1。但是，中间 GAG中的所有裂解部位MA/CA裂解位点对不足的处理最敏感 影响感染力。因此，我们承诺开发可在高度使用的生化测定 小分子抑制剂的吞吐量筛选。该屏幕（SRI筛选的775,000种化合物）现在 以最活跃的化合物为单位，其IC50从1 um开始完成。这些化合物已经 被证明不是HIV-1蛋白酶抑制剂。在此应用程序中，我们提出了四个目标。首先，我们将测试 使用我们的两个基于基于凝胶的测定法，其中最活跃的化合物包括 靶MA/CA底物和作为内部对照A可以被HIV-1蛋白酶切割但 这不应该是抑制剂的靶标。其次，我们将使用新颖的EC50测量病毒复制 测定将大大提高我们检测特定抑制的敏感性。第三，我们将测量A的EC50 来自不同进化枝的传播/创始人病毒和病毒的面板，以提供初步评估 序列变异性对灵敏度的影响。第四，对于那些具有足够治疗的化合物 索引我们将开始选择阻力以验证MA/CA目标。通过这项努力，我们将确定最大的 在这个新屏幕中有希望的命中，然后将来可以用作结构研究的基础 sar。