Developing Small Molecule Screens for Vif-APOBEC3 antagonists

开发 Vif-APOBEC3 拮抗剂的小分子筛选

• 批准号: 9058985
• 负责人: John D Gross
• 金额: $ 19.81万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9058985
• 关键词: 26S proteasome; APOCEC3G gene; Anti-Retroviral Agents; Binding; Biochemical; Biological Assay; CD4 Positive T Lymphocytes; CUL5 gene; Cell Culture Techniques; Cell Membrane Permeability; Cells; Chemicals; Clinic; Collaborations; Complex; Core-Binding Factor; Crystallography; Cullin Proteins; Data; Development; Drug Design; Drug Targeting; Enzymes; Family; Family member; Figs - dietary; Fluorescein-5-isothiocyanate; Fluorescence Resonance Energy Transfer; Funding; Genetic Transcription; HIV; Hand; Health; Human; Immune System Diseases; Immunity; Infection; Integration Host Factors; Lead; Libraries; Life; Ligase; Methods; Molecular Probes; Molecular Weight; Monitor; Mutation; Persons; Polyubiquitin; Polyubiquitination; Positioning Attribute; Proteins; Reagent; Recombinants; Reporting; Research; Role; Secondary to; Seeds; Series; Staging; Staining method; Stains; Structure; System; Testing; Time; Toxic effect; Ubiquitin Like Proteins; United States; Viral; Virus; Virus Replication; base; biophysical techniques; cofactor; drug use screening; high throughput screening; inhibitor/antagonist; multicatalytic endopeptidase complex; next generation; novel; programs; reconstitution; resistance mutation; scaffold; screening; small molecule; ubiquitin ligase; ubiquitin-protein ligase; vif Gene Products

 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.

 描述（由申请人提供）：目前正在进行一项重大努力，以确定由HIV增选的宿主复合物，以开发不易受逃逸突变影响的下一代抗逆转录病毒疗法。一个有前途的药物靶点是HIV辅助蛋白Vif，其与由Cullin-Ring Ligase 5（CRL 5）和核心结合因子β（CBF β）组成的宿主泛素E3连接酶复合物结合，以下调APOBEC 3家族的限制性因子。我们已经重建了Vif-E3泛素连接酶活性的重组组件，并建议开发一系列的初级和二级筛选，以发现Vif-E3连接酶抑制剂。在目标1中，我们将开发Vif-E3连接酶抑制剂的基于活性的高通量筛选。这 筛选消除了查询复合物中任何给定蛋白质相互作用对的潜在偏差， 最大化抑制剂发现的机会。在目标2中，我们将开发一套用于跟踪命中的二级测定，包括正交二级高通量测定、使用相关细胞E3确定命中选择性的计数器筛选、报告靶结合的生物物理测定（如SPR和NMR）、作用机制研究和评价对病毒感染性影响的测定。与以前的筛选Vif抑制剂不同，Vif抑制剂是基于细胞的，我们的方法采用重组成分，允许发现靶向特定生化系统的抑制剂。这将有助于使用基于结构的药物设计范例进行靶点识别和先导物优化。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。在这些筛选中发现的命中可能为下一代抗逆转录病毒疗法提供有希望的线索。或者，它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CRL 5在自身免疫疾病中的作用。