Discovery of Chemical Probes for RNA-Binding Protein Host Defense Factors

 描述（由申请人提供）：本修订提案是对项目公告PA-12-060的回应，该项目公告标题为“体内化学探针发现的验证命中请求”（R 01）。本研究的目的是合成和选择新型的化学支架作为APOBEC 3G（A3 G）与抑制性RNA结合的拮抗剂。这些将作为分子探针，以确定RNA结合A3 G在阻止宿主防御HIV感染中的作用。如果A3 G能够逃避被称为Vif（在晚期感染期间产生）的HIV蛋白的破坏，并且如果它与病毒颗粒一起包装，从而进入病毒粒子的细胞，那么A3 G在宿主防御HIV方面是有效的。在感染之前预先存在于细胞中的大量A3 G在很大程度上是惰性的，因为其活性通过其与细胞RNA的核糖核蛋白复合物的非选择性形成而严重减弱。RNA与A3 G的N-末端结合，并变构地抑制C-末端的ssDNA结合和脱氨酶结构域。OyaGen，Inc的高通量筛选已经鉴定了三种经验证的化学支架，其具有以下能力：（1）拮抗RNA与A3 G的结合和（2）在细胞中抢先激活A3 G，达到抑制进入的HIV复制的程度。在拟议的研究中，OyaGen，Inc和Sanford/Burnham将合作：（1）应用合理设计合成三种经验证的A3 G选择性抗病毒化合物的化学修饰，（2）选择具有增强活性的化合物作为A3 G：RNA结合和抗病毒活性的拮抗剂，（3）基于增强的A3 G DNA诱变活性和对细胞A3 G-RNA复合物的选择性选择最终探针，以及。(4)根据体外ADME/T和小鼠体内PK对化合物进行分类，以选择具有药物样特征的化合物。这些探针将带来重大的范式变化，因为它们将使研究和监测APOBEC 3G的作用机制成为可能，因为它在活病毒感染期间自然地在人类细胞中表达，并且将来在动物模型中表达。

 DESCRIPTION (provided by applicant): This revised proposal is in response to the Program Announcement PA-12-060 entitled 'Solicitation of Validated Hits for the Discovery of in vivo Chemical Probes' (R01). The goal of this proposal is to synthesize and select novel chemical scaffolds as antagonists of APOBEC3G (A3G) binding to inhibitory RNA. These will serve as molecular probes to determine the role that RNA binding to A3G has in preventing host defense against an HIV infection. A3G is effective in host defense against HIV if it can evade destruction by the HIV protein known as Vif (made during late infection) and if it becomes packaged with viral particles and thereby enters cells with virions. The bulk of A3G preexisting in cells prior t an infection is largely inert as its activity is severely attenuated through its nonselective formation of ribonucleoprotein complexes with cellular RNAs. RNA binds to the N-terminus of A3G and allosterically inhibits the ssDNA binding and deaminase domain in the C-terminus. High throughput screening by OyaGen, Inc has identified three validated chemical scaffolds for their ability to: (1) antagonize RNA binding to A3G and (2) preemptively activate A3G in cells to the extent that incoming HIV replication is inhibited. In the proposed research OyaGen, Inc and Sanford/Burnham will collaborate to: (1) apply rational design to synthesize chemical modifications of three validated A3G- selective antiviral compounds, (2) select those with enhanced activity as antagonists of A3G:RNA binding and antiviral activity, (3) select the ultimate probe(s) based on enhanced activation of A3G DNA mutagenic activity and selectivity for cellular A3G-RNA complexes, and. (4) triage compounds to select those with drug-like characteristics based on in vitro ADME/T and in vivo PK in mice. These probes will usher in a significant paradigm change as they will enable studies and monitoring of the mechanism of action of APOBEC3G as it naturally is expressed in human cells during a live virus infection and, in the future, in animal models.