Biological Activity of Lead Compounds Targeting HIV-1 TAR RNA

描述（由申请人提供）：本提案的目的是检测，同样重要的是合理化，靶向HIV LTR中反式激活反应元件（TAR）RNA的抗HIV先导化合物的细胞活性和特异性。在相关研究中，两位主要研究者使用新的“基于动力学”的虚拟屏幕来识别导联。这些研究使用各种生物物理技术以前所未有的深度和广度在体外建立了先导化合物的结合和活性特征，但它们不包括识别可能进一步开发成抗HIV治疗剂的有前景的先导化合物所需的关键生物测定。通过对这些“高度生物药理学特征”的先导化合物进行严格的毒性以及针对HIV-1和密切相关的HIV-2的生物测定，我们建议（i）鉴定具有体内活性的有希望的先导物，其可以在开发抗HIV疗法中进行进一步优化，和（ii）描绘RNA的生物物理性质之间的联系，小分子结合，其可以在体外以半高通量方式获得，以及生物活性和特异性，其对于实验测定来说要麻烦得多。我们将检验“RNA靶向特异性”是细胞活性和毒性的主要决定因素以及具有体外活性的RNA靶向化合物未能表现出体内抗病毒活性的主要原因的假设。我们将开发用于定量RNA靶向特异性的体外测定方法 并使用新的特异性度量来缩小进行细胞测定的化合物，以及测试具有精确选择性的靶向柔性RNA系统的新策略，该策略基于区域特异性拥挤阳离子基团。通过弥合深入的生物物理特性和生物测定之间的鸿沟，我们的研究将为预测理解RNA靶向奠定基础，RNA靶向可用于合理鉴定具有抗逆转录病毒活性的化合物。 公共卫生相关性：拟议的研究将对靶向HIV-1 LTR中的反式激活反应元件（TAR）的生物药理学充分表征的先导化合物进行毒性和生物学测定。这些研究将大大加快寻找和测试针对新RNA成分的新型治疗药物的步伐，从而进一步抑制艾滋病毒复制和耐药性的速度，并有助于扩大可用的治疗选择。

DESCRIPTION (provided by applicant): The goal of this proposal is to test, and equally importantly rationalize, the cellular activity and specificity of anti-HIV lead compounds that targt the transactivation response element (TAR) RNA in the HIV LTR. The leads are identified in related studies by the two principal investigators using a new 'dynamics-based' virtual screen. These studies establish the binding and activity characteristic of lead compounds in vitro with unprecedented depth and breadth using an assortment of biophysical techniques, but they do not include the key biological assays needed to identify promising leads that could be developed further into anti-HIV therapeutics. By subjecting a strategically chosen panel of these 'highly biophysically characterized' lead compounds to rigorous toxicity as well as biological assays against HIV-1 and the closely related HIV-2, we propose to (i) identify promising leads with in vivo activity that may be subjected to further optimization in the developed of anti-HIV therapeutics and (ii) delineate the link between the biophysical properties of RNA-small molecule binding, which can be obtained in a semi- high throughput manner in vitro, and biological activity and specificity, which is far more cumbersome to experimentally assay. We will test the hypothesis that 'RNA-targeting specificity' is a major determinant of cellular activiy and toxicity and the main reason RNA-targeting compounds with in vitro activity fail to exhibit in vivo anti-viral activity. We will develop in vitro assays for quantifying RNA-targeting specificity and use a new specificity metric to narrow down compounds that are subjected to cellular assays as well as test a new strategy for targeting flexible RNA systems with exquisite selectivity that is based on regiospecifically crowding cationic groups. By bridging the divide between in-depth biophysical properties and biological assays, our studies will lay the foundations for a predictive understanding of RNA- targeting that can be used to rationally identify compounds with anti-retroviral activity. PUBLIC HEALTH RELEVANCE: The proposed research will subject biophysically well-characterized lead compounds that target the transactivation response element (TAR) in the HIV-1 LTR to toxicity and biological assays. The studies will significantly accelerate the pace of searching and testing for novel therapeutic agents that target new RNA components and thereby further suppress the rate of HIV replication and resistance, as well as help widen the treatment options available.