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

描述(申请人提供)：APOBEC3G(A3G)是一种在人类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，A3G DNA对新生单链前病毒DNA的突变活性可以抑制病毒复制，但最近的研究表明，在许可细胞中，A3G突变活性可能有利于病毒并促进耐药株的出现。这项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紧迫问题的新研究工具，即内源性表达的A3G脱氨酶活性是否可以被调节到超过其具有抗病毒活性所需的诱变阈值。这是一个相关的问题，因为在允许的细胞中，A3G通过与细胞RNA的相互作用几乎完全“关闭”，这种低水平的活动可能对病毒有利。初步研究已经确定了通过将RNA从酶上解离来激活A3G脱氨酶活性的化合物，并且这些化合物在A3G以高分子量复合体的形式隔离的许可细胞中具有剂量依赖的抗病毒作用。这些化合物是同类化合物中的第一个，表明在活细胞中对A3G的RNA抑制是可逆的。今年R21的具体目标是：(1)利用组装的独特小分子文库对A3G激活剂进行高通量筛选和构效关系分析，以确定在单轮感染性检测中具有低细胞毒性和抗病毒活性的靶点；(2)确定A3G激活剂抑制A3G与RNA结合的特异性和选择性，并定量它们对病毒DNA载量和突变频率的影响；(3)通过PBMC和活病毒7天传播性试验证实验证的HITS的抗病毒活性和低细胞毒性。通过这一提议确定的化合物实现了将一类新化合物表征为“独特的研究分子探针”的目标，这些化合物为攻击病毒耐药性提供了新的治疗目标和药物发现机会，同时满足了对解决该领域争议的新研究工具的未得到满足的需求。 公共卫生相关性：该提案是对美国国立卫生研究院探索性发展研究资助项目PA-10-069项目公告的回应。该建议的最终目标是寻找一种或多种新型抗病毒化合物用于治疗开发，这些化合物具有纳摩尔疗效和低毒，其作用机制是将APOBEC3G(A3G)从与宿主细胞RNA形成的聚集体中释放出来。这种‘A3G激活剂’已经通过使用创新的猝灭FRET试验对HTS进行了初步高通量筛查而被识别和验证。通过拟议的研究确定的化合物也将用于解决对新研究试剂的未得到满足的需求，以解决关于3G诱变活性是否促进宿主防御或有利于病毒的争议。这一提议具有创新性，因为它寻求了降低病毒传染性和出现病毒耐药性的非传统解决方案。“这项拟议研究的长期目标是确定新的化合物，这些化合物可以开发成治疗或预防艾滋病毒/艾滋病的药物。”

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'.