Development of a Novel HIV/AIDS Therapeutic Activating the APOBEC3G Host Defense

开发一种激活 APOBEC3G 宿主防御的新型 HIV/AIDS 疗法

• 批准号: 8263387
• 负责人: Harold C Smith
• 金额: $ 21万
• 依托单位: OYAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8263387
• 关键词: AIDS/HIV problem; Address; Adverse effects; Antiviral Agents; Binding; Biological Assay; Cell Line; Cells; Chemistry; Communicable Diseases; Complex; Cytoplasm; DNA; Data; Deaminase; Development; Dose; Drug resistance; Drug usage; EMSA; Electrophoretic Mobility Shift Assay; End Point Assay; Enzymes; Evaluation; Exhibits; Failure; Fee-for-Service Plans; Fluorescence Resonance Energy Transfer; Frequencies; Genome; Goals; Growth; HIV; Host Defense; Human; In Vitro; Infection; Inhibitory Concentration 50; Lead; Leukocytes; Libraries; Life; Mitogens; Molecular Probes; Mutagenesis; Mutation; NIH Program Announcements; Peripheral Blood Mononuclear Cell; Prevention; Probability; Process; Promega; Property; Proteins; RNA; RNA Binding; RNA-Binding Proteins; RNA-Directed DNA Polymerase; Reagent; Reporting; Research; Research Project Grants; Solutions; Specificity; Structure; Structure-Activity Relationship; Testing; Therapeutic; Time; Toxic effect; United States National Institutes of Health; Validation; Viral; Viral Proteins; Virus; base; cytotoxicity; design; drug discovery; drug resistant virus; efficacy testing; high throughput screening; in vitro activity; innovation; knockout gene; molecular mass; new therapeutic target; novel; prevent; programs; protein aggregate; public health relevance; resistant strain; response; small molecule; small molecule libraries; therapeutic development; therapeutic target; tool; viral DNA; viral RNA; viral resistance

DESCRIPTION (provided by applicant): APOBEC3G (A3G) is a protein expressed in human cells that serves as an antiviral host-defense factor. While the majority of studies have suggested that A3G DNA mutagenic activity on nascent single stranded proviral DNA inhibits viral replication, recent studies suggested that circumstances might exist in permissive cells where A3G mutagenic activity may benefit the virus and promote the emergence of drug-resistant strains. The proposed research will identify small molecules for therapeutic development that are also novel research tools for addressing the urgent question of whether endogenously expressed A3G deaminase activity can be modulated to exceed a mutagenic threshold necessary for it to have antiviral activity. This is a relevant question because, in permissive cells, A3G is almost entirely 'switched off' through its interaction with cellular RNA and this low level of activity may benefit the virus. Preliminary studies have identified compounds that activate A3G deaminase activity by dissociating RNA from the enzyme and these compounds had a dose- dependent antiviral effect in permissive cells where A3G was sequestered as high molecular mass complexes. These compounds are first in their class and show that RNA inhibition of A3G is reversible in living cells. The Specific Aims for this two year R21 are: (1) conduct high throughput screening and structure activity relationship analyses for A3G activators using a unique small molecule library assembled to identify target-select hits with low cytotoxicity and antiviral activity in single round infectivity assays, (2) determine the specificity and selectivity of A3G activators for inhibiting RNA binding to A3G and quantify their effect on viral DNA load and mutagenic frequency and (3) confirm the antiviral activity and low cytotoxicity of validated hits using PBMC and live virus in 7-day spreading infectivity assays. Compounds identified through this proposal achieve the goal of characterizing a novel class of compounds 'as unique research molecular probes' and that offer a new therapeutic target and drug discovery opportunity for attacking viral resistance while satisfying an unmet need for novel research tools to address controversies in the field. PUBLIC HEALTH RELEVANCE: This proposal is in response to Program Announcement PA-10-069, NIH Exploratory Developmental Research Grant Program. The ultimate goal of this proposal is to identify one or more novel antiviral compounds for therapeutic development that have nanomolar efficacy and low toxicity whose mechanism of action is to liberate APOBEC3G (A3G) from aggregates that form with host cell RNA. Such 'A3G Activators' have been identified and validated through the preliminary high throughput screening HTS with an innovative quenched FRET assay. Compounds identified through the proposed research will also serve to address an unmet need for novel research reagents to address controversy over whether A3G mutagenic activity promotes host defense or benefits the virus. This proposal is innovative because it seeks unconventional solutions for reducing viral infectivity and the emergence of viral resistance. 'The long term goal of the proposed research is to identify novel compounds that can be developed into drugs used in the treatment or prevention of HIV/AIDS'.

描述（由申请人提供）：APOBEC 3G（A3 G）是一种在人细胞中表达的蛋白质，作为抗病毒宿主防御因子。虽然大多数研究表明A3 G DNA对新生单链前病毒DNA的诱变活性抑制病毒复制，但最近的研究表明，在允许细胞中可能存在A3 G诱变活性可能有益于病毒并促进耐药菌株的出现的情况。拟议的研究将确定用于治疗开发的小分子，这些小分子也是解决内源性表达的A3 G脱氨酶活性是否可以被调节以超过其具有抗病毒活性所需的诱变阈值的紧迫问题的新研究工具。这是一个相关的问题，因为在允许的细胞中，A3 G通过与细胞RNA的相互作用几乎完全“关闭”，这种低水平的活性可能有利于病毒。初步研究已经鉴定了通过从酶解离RNA来激活A3 G脱氨酶活性的化合物，并且这些化合物在A3 G作为高分子量复合物被隔离的允许细胞中具有剂量依赖性抗病毒作用。这些化合物是同类中的第一个，表明A3 G的RNA抑制在活细胞中是可逆的。这两年R21的具体目标是：（1）使用组装的独特小分子文库对A3 G激活剂进行高通量筛选和结构活性关系分析，以在单轮感染性测定中鉴定具有低细胞毒性和抗病毒活性的靶选择命中，（2）确定A3 G激活剂抑制RNA与A3 G结合的特异性和选择性，并定量其对病毒DNA载量和诱变频率的影响，以及（3）在7天传播感染性试验中，使用PBMC和活病毒确认经验证的命中的抗病毒活性和低细胞毒性。通过该提议鉴定的化合物实现了将一类新型化合物表征为“独特的研究分子探针”的目标，并且为攻击病毒抗性提供了新的治疗靶标和药物发现机会，同时满足了对新的研究工具的未满足的需求，以解决该领域的争议。 公共卫生相关性：本提案是对计划公告PA-10-069，NIH探索性发展研究资助计划的回应。该提议的最终目标是鉴定一种或多种用于治疗开发的新型抗病毒化合物，其具有纳摩尔功效和低毒性，其作用机制是从与宿主细胞RNA形成的聚集体中释放APOBEC 3G（A3 G）。这种“A3 G激活剂”已经通过具有创新的淬灭FRET测定的初步高通量筛选HTS来鉴定和验证。通过拟议的研究确定的化合物也将有助于解决对新型研究试剂的未满足需求，以解决关于A3 G诱变活性是否促进宿主防御或有益于病毒的争议。这项提议是创新的，因为它寻求非传统的解决方案，以减少病毒感染性和病毒耐药性的出现。“拟议研究的长期目标是确定可以开发成用于治疗或预防艾滋病毒/艾滋病的药物的新化合物。