Identifying A New Class of HIV Maturation Inhibitors

摘要 有效的抗艾滋病毒药物一直集中在有限数量的靶点上。虽然这些药物目前高度 出于两个原因，有必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，抗药性可能会成为一个问题；因此 现在开发新的靶点和抑制剂为开发新的 未来的毒品。其次，抑制物代表化学探针，可以补充遗传方法在 研究生物现象的术语。因此，非药物的抑制剂仍然具有内在的工具价值。 了解复杂的生物系统。我们之前已经证明了MA/CA蛋白水解酶的切割位点 在HIV-1中，Gag蛋白必须被切割到接近完成，才能形成具有感染性的病毒颗粒。连 10%的这个位点的欠切割会毒害组装过程，可能是因为一些未切割的MA/CA 融合蛋白参与衣壳的形成并毒化这一高度调控的过程。Bevirimat形成了一个 考虑特定切割位点的抑制剂的概念基础，在这种情况下是CA/SP1。然而，在 到目前为止，GAG中的所有裂解位点MA/CA裂解位点对欠加工最敏感 影响传染性。出于这个原因，我们致力于开发一种生化分析方法，这种方法可以用于 小分子抑制剂的吞吐量筛选。这一筛选(SRI筛选了775,000种化合物)现在 最活跃的化合物的IC50从1微米开始。这些化合物已经 已被证明不是HIV-1蛋白酶抑制剂。在本申请中，我们提出了四个目标。首先，我们将测试 使用我们的双底物凝胶分析在二次筛选中最有活性的化合物，它包括 靶向MA/CA底物和作为内对照的可被HIV-1蛋白酶切割的底物，但 这不应该是抑制剂的目标。其次，我们将使用一种新的方法来测量病毒复制的EC50 这将大大提高我们检测特定抑制的敏感度。第三，我们将测量EC50 传播/创始病毒和来自不同分支的病毒的小组，以提供对 序列变异性对敏感性的影响。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指数，以验证MA/CA目标。通过这一努力，我们将确定最多 在这个新的屏幕上有希望的点击，然后可以在未来用作结构研究的基础和 萨尔。

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.