Identifying A New Class of HIV Maturation Inhibitors

摘要 抗艾滋病毒药物的作用集中在有限的靶点上。虽然这些药物目前在 由于两个原因，需要继续开发新的目标。首先，有可能在 在未来几十年治疗感染者的过程中，耐药性可能成为一个问题;因此， 开发新的靶标和抑制剂现在为开发新的药物提供了理论基础。 未来的毒品第二，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，不是药物的抑制剂仍然具有作为工具的内在价值， 了解复杂的生物系统。我们以前已经表明，MA/CA蛋白酶切割位点 在HIV-1 Gag蛋白中，必须切割到接近完全，以允许形成感染性病毒颗粒。甚至 该位点的10%切割不足会使组装过程中毒，这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使这一高度调节的过程中毒。贝韦瑞马特形成了 概念基础的思考抑制剂的具体裂解位点，在这种情况下CA/SP1。然而， 在Gag中的所有切割位点中，MA/CA切割位点对加工不足最敏感， 影响传染性。为此，我们着手开发一种生化测定法，可用于高浓度的 小分子抑制剂的通量筛选。该筛选（由SRI筛选的775，000种化合物）现在已经完成。 用IC 50从1 μ M开始的最具活性的化合物完成。这些化合物已经 它不是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 使用我们的基于双底物凝胶的测定法进行二次筛选， 靶MA/CA底物和作为内部对照的可被HIV-1蛋白酶切割但 其不应该是抑制剂的靶标。其次，我们将使用一种新的方法测量病毒复制的EC 50。 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EC 50， 传播/创始者病毒和来自不同进化枝的病毒的小组，以提供对 序列变异性对灵敏度的影响。第四，对于那些有足够治疗效果的化合物， 我们将开始选择耐药性指标，以验证MA/CA目标。通过这一努力，我们将确定最 在这个新的屏幕上有希望的命中，然后可以在未来用作结构研究的基础， 特别行政区

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.