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PA-12-060号计划公告的回应，该公告标题为“征集发现体内化学探针的确认命中”(R01)。本研究的目的是合成和筛选新型化学支架作为APOBEC3G(A3G)与抑制性RNA结合的拮抗剂。这些将作为分子探针来确定与A3G结合的RNA在防止宿主防御艾滋病毒感染方面所起的作用。如果A3G能够逃避被称为Vif的HIV蛋白质的破坏(在感染后期产生)，并且它被病毒颗粒包裹，从而进入带有病毒粒子的细胞，那么A3G在抵抗HIV方面是有效的。在感染前预先存在于细胞中的大部分A3G在很大程度上是惰性的，因为它的活性因其与细胞RNA非选择性地形成核糖核蛋白复合体而严重减弱。RNA与A3G的N-末端结合，变构抑制C-末端的单链DNA结合和脱氨酶结构域。OyaGen公司的高通量筛选已经确定了三种经过验证的化学支架，它们具有以下能力：(1)拮抗与A3G的RNA结合；(2)在细胞中先发制人地激活A3G，从而抑制传入的艾滋病毒复制。在拟议的研究中，OyaGen，Inc.和Sanford/Burnham将合作：(1)应用合理的设计合成三种有效的A3G选择性抗病毒化合物的化学修饰，(2)选择那些活性增强的化合物作为A3G的拮抗剂：RNA结合和抗病毒活性，(3)基于增强的A3GDNA突变活性和对细胞A3G-RNA复合体的选择性选择最终探针(S)，以及。(4)根据体外ADME/T和体内PK筛选出具有类药物特征的化合物。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APOBEC3G的作用机制成为可能，因为在活体病毒感染期间，APOBEC3G在人类细胞中自然表达，未来将在动物模型中表达。

 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.