Developing Small Molecule Screens for Vif-APOBEC3 antagonists

 描述(由申请人提供)：正在进行一项重大努力，以确定艾滋病毒增选的宿主复合体，以便开发不太容易逃脱突变的下一代抗逆转录病毒疗法。一个有前景的药物靶点是HIV辅助蛋白Vif，它与宿主泛素E3连接酶复合体(CRL5)和核心结合因子β(CBF？)共同下调APOBEC3限制因子家族。我们已经从重组组分中重组了Vif-E3泛素连接酶的活性，并建议开发一系列初级和二级筛选来发现Vif-E3连接酶抑制剂。在目标1中，我们将开发一种基于活性的高通量筛选Vif-E3连接酶抑制剂。这 Screen消除了在查询复合体中的任何给定蛋白质相互作用对时的潜在偏见， 最大限度地增加发现抑制剂的机会。在目标2中，我们将开发一套跟踪HITS的二次检测方法，包括一种正交的二次高通量检测方法，使用相关的细胞E3来确定HITS的选择性的计数器筛选，报告靶标结合的生物物理方法，如SPR和核磁共振，作用机制研究，以及评估对病毒感染性影响的方法。与以前基于细胞的Vif抑制剂筛选不同，我们的方法使用重组组件，允许发现针对特定生化系统的抑制剂。这将有助于目标识别和使用基于结构的药物设计范例进行优化。这些筛选可以应用于其他利用CRL机制下调限制因子的病毒辅助蛋白。在这些筛查中发现的热门药物可能为下一代抗逆转录病毒疗法提供有希望的线索。或者，他们可以提供分子探针来探索Vif和A3家族成员之间的不同相互作用，或者CRL5在自身免疫性疾病中的作用。

 DESCRIPTION (provided by applicant): A major effort is underway to define host complexes co-opted by HIV in order to develop next generation anti- retroviral therapies that are less susceptible to escape mutations. A promising drug target is the HIV accessory protein Vif, which coopts a host ubiquitin E3 ligase complex consisting Cullin-Ring Ligase 5 (CRL5) and core binding factor beta (CBFß) to downregulate the APOBEC3 family of restriction factors. We have reconstituted the Vif-E3 ubiquitin ligase activity from recombinant components and propose to develop a series of primary and secondary screens to discover Vif-E3 ligase inhibitors. In Aim 1 we will develop an activity-based high- throughput screen for inhibitors of the Vif-E3 ligase. This screen eliminates potential bias in querying any given pair of protein interactions in the complex, maximizing the chance for inhibitor discovery. In Aim 2 we will develop a suite of secondary assays for following up on hits, including an orthogonal secondary high- throughput assay, a counter screen to determine selectivity of hits using a related cellular E3, biophysical assays such as SPR and NMR to report on target binding, mechanism of action studies, and assays to evaluate effects on viral infectivity. Unlike previous screens for Vif inhibitors, which were cell based, our approach employs recombinant components, allowing the discovery of inhibitors that target a defined biochemical system. This will facilitate target identification and lead optimizatin using the structure-based drug design paradigm. These screens could be applied to other viral accessory proteins that make use of the CRL machinery to downregulate restriction factors. Hits discovered in these screens could provide promising leads for next generation anti-retroviral therapies. Alternatively, they may provide molecular probes to explore diverse interactions between Vif and A3 family members or the role of CRL5 in auto-immune disease.