Biological Activity of Lead Compounds Targeting HIV-1 TAR RNA

描述(由申请人提供)：本提案的目标是测试，同样重要的是使抗HIV先导化合物的细胞活性和特异性合理化，这些化合物以HIV LTR中的反式激活反应元件(TAR)RNA为靶标。两位主要研究人员在相关研究中使用了一种新的“基于动力学”的虚拟屏幕来识别这些线索。这些研究利用各种生物物理技术以前所未有的深度和广度确立了先导化合物在体外的结合和活性特征，但它们不包括确定可进一步开发为抗艾滋病毒疗法的有希望的先导化合物所需的关键生物分析方法。通过对一组经过精心挑选的“具有高度生物物理特性”的先导化合物进行严格毒性以及针对HIV-1和密切相关的HIV-2的生物测试，我们建议：(I)确定在抗HIV治疗学开发中可能有待进一步优化的具有体内活性的有希望的先导化合物；(Ii)勾勒出可在体外以半高通量方式获得的RNA-小分子结合的生物物理性质，与生物活性和特异性之间的联系，后者对实验分析要麻烦得多。我们将检验这一假设，即RNA靶向特异性是细胞活性和毒性的主要决定因素，也是具有体外活性的RNA靶向化合物在体内未能表现出抗病毒活性的主要原因。我们将开发定量RNA靶向特异性的体外检测方法。 并使用新的特异性指标来缩小接受细胞分析的化合物的范围，以及测试一种新的策略，以基于区域特定的阳离子基团的精确选择性来靶向灵活的RNA系统。通过弥合深入的生物物理性质和生物检测之间的鸿沟，我们的研究将为预测理解RNA靶向奠定基础，该靶向可用于合理地识别具有抗逆转录病毒活性的化合物。

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.