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工作来定义 HIV 所选择的宿主复合物，以开发不易受到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，它与由 Cullin-Ring Ligase 5 (CRL5) 和核心结合因子 b​​eta (CBFß) 组成的宿主泛素 E3 连接酶复合物共同下调 APOBEC3 限制因子家族。我们从重组成分中重建了 Vif-E3 泛素连接酶活性，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 Vif-E3 连接酶抑制剂的高通量筛选。这 筛选消除了查询复合物中任何给定的一对蛋白质相互作用时的潜在偏差， 最大限度地提高发现抑制剂的机会。在目标 2 中，我们将开发一套用于跟踪命中的二次测定，包括正交二次高通量测定、使用相关细胞 E3 确定命中选择性的计数器筛选、报告靶标结合的生物物理测定（例如 SPR 和 NMR）、作用机制研究以及评估对病毒感染性影响的测定。与之前基于细胞的 Vif 抑制剂筛选不同，我们的方法采用重组成分，从而能够发现针对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式进行靶标识别和先导化合物优化。这些筛选可应用于其他利用 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以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 A​​3 家族成员之间的不同相互作用或 CRL5 在自身免疫性疾病中的作用。