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）颗粒组装的新型有效抑制剂。目前对感染艾滋病毒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药物，但不断出现的多重耐药病毒株需要发现针对新的艾滋病毒靶点的新型药物。我们将重点关注衣壳蛋白（CA）， HIV病毒粒子的主要成分：它的二聚化是病毒粒子组装的第一步。目前还没有针对这一靶点的抗hiv药物被临床开发出来。然而，测量衣壳多聚的方法最近被用于鉴定阻断病毒组装和HIV复制的原理证明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毒株中。我们设计了一种超高通量筛选试验（uHTS），以及伴随筛选和反筛选来鉴定衣壳组装的有效和选择性抑制剂。通过将能量转移生化筛选与hiv感染细胞的二级分析相结合，我们将能够发现高亲和力的新化合物。我们将依次i)在1536孔微孔板格式中进行初级uHTS筛选，以在已有的350,000个化合物MPLCN文库中鉴定HIV CA二聚化抑制剂ii)分类非特异性干扰特异性或不相关蛋白-蛋白测定的化合物，并消除用于研究HIV影响的细胞的命中细胞毒性ii)评估命中化合物对HIV感染细胞的抑制CA产生和HIV传染性的能力。iv)验证受抑制剂影响的生命周期阶段，v)调查我们的化合物抑制的实验室适应的HIV-1和HIV-2株和临床株的谱。为了证明交替标记的CA蛋白的二聚化作用，我们设计了一套互补和正交试验。初步的TR-FRET检测将在384孔微滴板上进行优化，并在模型文库（例如LOPAC）的中试筛选中进行验证，然后运行大型MPLCN筛选，使用NYAD-1肽和PF-74小化合物衣壳抑制剂作为参考。我们的HIV ca特异性AlphaScreen将作为辅助试验来帮助表征命中化合物，以进一步进行生物学验证。几个反掩护将有助于消除假阳性命中。CA特异性抑制剂将通过其抑制CA蛋白n端和c端部分相互作用的能力来区分，使用基于CA的检测。基于次级细胞的检测HIV传染性检测将用于表征HIV复制的非细胞毒性特异性高亲和力抑制剂。将与其他已知的抗艾滋病毒化合物（包括AZT、Efavirenz和Saquinavir）进行比较，确定命中目标的hiv复制周期的阶段。我们的目标是为科学界提供可自由获取的可靠的检测方法和有效的、在纳摩尔浓度下具有活性的多种作用的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.