Targeting HIV Capsid Assembly

描述（由申请人提供）：我们将启用超高通量筛选策略，以鉴定人类免疫缺陷病毒（HIV）颗粒组装的新型强效抑制剂。目前对艾滋病毒感染的艾滋病患者的治疗是基于针对病毒复制周期早期和晚期几个关键步骤的药物组合。虽然已经开发出了高效的药物，但多重耐药病毒株的不断出现需要发现针对新的HIV靶点的新药物类别。我们将集中在衣壳蛋白（CA），HIV病毒粒子的主要组成部分：它的二聚化是病毒颗粒组装的第一步。目前临床上尚未开发出针对该目标的抗艾滋病毒药物。然而，测量衣壳多聚化的方法最近已用于鉴定阻断病毒组装和HIV复制的原理验证肽和小化合物抑制剂，包括在对靶向病毒酶的药物具有抗性的HIV菌株中。我们设计了一种超高重复率筛选-适合测定（uHTS），以及伴随筛选和反筛选，以鉴定衣壳组装的有效和选择性抑制剂。通过将能量转移生物化学筛选与HIV感染细胞中的二级检测相结合，我们将能够发现具有高亲和力的新型化合物。我们将相继i）在1，536孔微孔板格式中进行初级uHTS筛选，以鉴定在已有先例的350，000个化合物MPLCN文库中的HIV CA二聚化的抑制剂ii）分类非特异性干扰特异性或不相关蛋白质-蛋白质测定的化合物，并消除用于研究对HIV的影响的细胞的细胞毒性命中，ii）评估在HIV感染细胞上的命中化合物抑制CA产生和HIV感染性的能力，iv）验证受抑制剂影响的生命周期阶段，v）调查由我们的化合物抑制的HIV-1和HIV-2实验室适应和临床菌株的谱。为了证明使用交替标记的CA蛋白的二聚化，我们设计了一组互补和正交测定。将在384孔微量滴定板中优化初级TR-FRET测定，并在模型文库的中试筛选中验证（例如：LOPAC），然后使用NYAD-1肽和PF-74小化合物衣壳抑制剂作为参照进行大MPLCN筛选。我们的HIV CA特异性AlphaScreen将作为一种配套检测方法，帮助表征命中化合物，以进行进一步的生物学验证。几个反屏幕将用于消除假阳性命中。CA特异性抑制剂将通过其抑制CA蛋白的N-末端和C-末端部分之间的相互作用的能力来区分，通过使用基于CA的测定。基于细胞的二级试验HIV感染性试验将用于表征HIV复制的非细胞毒性特异性高亲和力抑制剂。通过与其他已知的抗HIV化合物（包括AZT、依法韦仑和沙奎那韦）进行比较，将确定命中所针对的HV复制周期阶段。我们的目标是为科学界提供可免费获得的强大检测方法和纳摩尔浓度的强效、双效HIV衣壳抑制剂。

DESCRIPTION (provided by applicant): We will enable an ultra high throughput screening strategy to identify novel potent inhibitors of Human Immunodeficiency Virus (HIV) particle assembly. Current treatments of HIV-infected AIDS patients are based on combinations of drugs that target several critical key steps in the early and late stages of the viral replication cycle. While highly effective drugs have been developed, continuous emergence of multi-resistant viral strains requires the discovery of new classes of drugs directed against novel HIV targets. We will focus on the capsid protein (CA), the main component of the HIV virion: its dimerization is the first step in assembly of the viral particle. No anti-HIV drugs have yet been clinically developed against this target. Methods to measure capsid multimerization have however been recently used to identify proof-of-principle peptides and small compound inhibitors which block viral assembly and HIV replication, including in HIV strains resistant to drugs targeting viral enzymes. We designed an ultra High-Throughput Screening-amenable assay (uHTS), and companion screens and counter-screens to identify potent and selective inhibitors of capsid assembly. By combining transfer-of-energy biochemical screens with secondary assays in HIV-infected cells, we will enable the discovery of novel compounds of high affinity. We will successively i) perform a primary uHTS screen in a 1,536 well microplate format to identify inhibitors of HIV CA dimerization in the well-precedented 350,000 compound MPLCN library ii) triage compounds interfering non-specifically with the specific or unrelated protein-protein assays, and eliminate hits cytotoxic for cells used for studying effects on HIV, ii) evaluate hit compounds on HIV-infected cells for their ability to inhibit CA production and HIV infectivity, iv) verify the stage of the life cycle affected by the inhibitors, v) survey the spectum of HIV-1 and HIV-2 laboratory-adapted and clinical strains inhibited by our compounds. To demonstrate dimerization using alternately tagged CA proteins, we designed a set of complementary and orthogonal assays. The primary TR-FRET assay will be optimized in 384-well microtiter plates and validated in pilot screens of model Libraries (e.g.: LOPAC) before running a large MPLCN screen, using as references the NYAD-1 peptide and the PF-74 small compound capsid inhibitors. Our HIV CA-specific AlphaScreen will serve as a companion assay to help characterize hit compounds for further biologic validation. Several counter-screens will serve to eliminate false-positive hits. CA-specific inhibitors will be differentiated by their capaity to inhibit interactions between the N-terminal and C-terminal portions of the CA protein, by using CA-based assays. Secondary cell-based assays HIV infectivity assays will be used to characterize non cytotoxic specific high-affinity inhibitors of HIV replication. The stage of the HV replication cycle targeted by the hits will be identified in comparison with other known anti-HIV compounds including AZT, Efavirenz and Saquinavir. Our goal is to provide the scientific community with freely accessible robust assays and potent, diversely acting HIV capsid inhibitors active in nanomolar concentrations.