Nucleic Acid Footprinting and development of small molecule antagonists

内部的努力已经导致了小分子微阵列的开发和实施，其上已经打印了20，000个小分子的文库。将荧光标记的RNA（长度为60个核苷酸）流过阵列，并将结合记录为荧光信号。重要的是，整个阵列可以在一周内探测，并且节省RNA量。使用这个系统，我们已经成功地确定了新的化学型，识别HIV-1的反式激活反应（TAR）的元素在生化检测，并抑制病毒复制的文化。药物化学成功地衍生出新的化学型，其结合位点通过NMR光谱法鉴定。这个“试点”项目已经成为扩大努力的基础，以小分子靶向调节RNAS。第二个正在研究的HIV顺式作用RNA是Rev反应元件（RRE），它对病毒基因组的核质转运很重要。RRE的茎环IIB（SLIIB）为HIV Rev蛋白提供了主要结合位点，最近对HIV-1基因组转录后修饰的分析揭示了RRE SLIIB中两个潜在的修饰位点。我们扩展了这些观察，以询问m6 A修饰的RNA是否为小分子识别提供了独特的信号。事实上，使用我们的小分子微阵列，除了识别两种RNA的第三类之外，还识别了识别非甲基化或m6 A-甲基化RRE SLIIB的化学型。了解m6 A修饰诱导的构象变化的性质与选择性小分子识别正在进行中。将小分子微阵列策略应用于KSHV PAN lncRNA的ENE元件，已经鉴定了识别三螺旋的化学型。生物学测试表明，这些化合物的子集是没有细胞毒性的，这使我们能够研究它们对KSHV复制的影响。出乎意料的是，一种化合物能够从潜伏期重新激活KSHV，其分子机制目前正在研究中。更重要的是，我们发现了一种从潜伏期激活KSV的新型化学型，这开辟了开发“踢杀”策略的可能性，从而激活的病毒可以被第二组靶向特定病毒酶的小分子靶向。正如稍后将概述的那样，实验室中的一个平行项目确实鉴定了靶向一种或多种HSHV核苷酸转移酶的小分子。我们还考虑了拮抗三螺旋形成可能被认为是一种治疗策略的选择。因此，研究了ENE的双链体版本，其揭示了额外的独特化学型。计划在不久的将来对这些化合物进行生物试验。该翻译项目与生物化学（SHAPE-MaP）和生物物理学方法（NMR，SAXS）相结合，提供有关ENE/小分子复合物的高分辨率信息，可用于基于结构的药物设计。由于我们设计的筛选策略需要将小分子共价连接到显微镜载玻片上，因此这有可能掩盖药效团。为了解决这一缺点，我们最近开发了一种基于溶液的筛选策略，该策略更适合于NCI分子靶标实验室（MTL）管理的大量小分子，特别是其大量天然产物提取物。利用登革病毒RNA基因组的茎环A（SLA）作为模型系统，我们已经开发了一种低成本，稳健和可重复的高通量热变性分析（HTS-Thermofluor），与新的化学型被确定从MTL收集的纯天然产物。我们还表明，同样的策略可以应用于MTL收集的天然产物提取物，这代表了一个独特的收集新的化学实体。SLA结合化学型的后续鉴定将通过LC/MS完成。250，000个分子的全筛选需要20 mg纯化RNA，并且通过其对调节RNA的NMR分析的承诺，实验室具有足够的高水平RNA生产和纯化能力。KSHV PAN的一个独特特征是它存在于细胞核、细胞质和纯化的病毒粒子中。与NCI ACVP的研究人员合作，我们已经完成了这些细胞区室中每个1200-nt PAN的结构分析，其目标是揭示病毒和/或细胞蛋白质对PAN占用的改变。这项研究最近发表在《核酸研究》上，扩展到绘制重组KSHV蛋白在体外转录的PAN RNA上的位点。

In-house efforts have led to development and implementation of a small molecule microarray onto which a library of 20,000 small molecules has been printed. Fluorescently-labeled RNAs ( 60 nucleotides in length) are flowed over the array, and binding is recorded as a fluorescent signal. Importantly, the entire array can be probed within one week, and is sparing on RNA amounts. Using this system, we have successfully identified novel chemotypes that recognize the HIV-1 trans-activation response (TAR) element in biochemical assays, and inhibit virus replication in culture. Medicinal chemistry was successful in deriving novel chemotypes, whose binding site was identified by NMR spectroscopy. This "pilot" project has been the basis for expanded efforts to target regulatory RNAS with small molecules. A second HIV cis-acting RNA under investigation is the Rev response element (RRE), which is important for nucleocytoplasmic transport of the viral genome. Stem-loop IIB (SLIIB) of the RRE provides the primary binding site for the HIV Rev protein, and recent analysis of post-transcriptional modification of the HIV-1 genome has revealed two potential sites of modification in RRE SLIIB. We extended such observations to ask whether m6A-modified RNA provides a unique signal for small molecule recognition. Indeed, using our small molecule microarray, chemotypes that recognized non-methylated or m6A-methylated RRE SLIIB were identified, in addition to a third class that recognized both RNAs. Understanding the nature of conformational changes induced by m6A modification with respect to selective small molecule recognition is underway. Applying the small molecule microarray strategy to the ENE element of KSHV PAN lncRNA has identified chemotypes that recognize the triple helix. Biological testing indicated that a subset of these compounds was not cytotoxic, which has allowed us to investigate their effect on KSHV replication. Unexpectedly, one compound was capable of reactivating KSHV from latency, and the molecular mechanism is presently under investigation. More importantly, our discovery of a novel chemotype that activates KSV from latency opens the possibility of developing a "kick-and-kill" strategy whereby activated virus can be targeted by a second set of small molecules targeted to specific viral enzymes. As will be outlined later, a parallel project in the laboratory has indeed identified small molecules that target one or more HSHV nucleotidyltransferases. We have also considered the option that antagonizing triple helix formation might be considered a therapeutic strategy. A duplex version of the ENE was therefore investigated, which has revealed additional, unique chemotypes. Biological testing of these compounds is planned for the near future. This translational project is combined with biochemical (SHAPE-MaP) and biophysical approaches (NMR, SAXS) to provide high resolution information on ENE/small molecule complexes that can be used for structure-based drug design. Since the screening strategy we have designed requires covalent linkage of small molecule to the microscope slide, this has the potential to mask the pharmacophore. In order to address this shortcoming, we have recently developed a solution-based screening strategy that is more amenable to the extensive collection of small molecules curated by the NCI Molecular Targets Laboratory (MTL), and in particular its vast collection of natural product extracts. Using stem-loop A (SLA) of the Dengue virus RNA genome as a model system, we have developed a low-cost, robust and reproducible high throughput thermal denaturation assay (HTS-Thermofluor), with which novel chemotypes were identified from the MTL collection of pure natural products. We have also shown that the same strategy can be applied to the MTL collection of natural product extracts, which represent a unique collection of novel chemical entities. Subsequent identification of SLA-binding chemotypes will be accomplished by LC/MS. A full screen of 250,000 molecules requires 20 mg of purified RNA, and, through its commitment to NMR analysis of regulatory RNAs, the laboratory has sufficient capacity for high level RNA production and purification. A unique feature of KSHV PAN is that it is present in the nucleus, cytoplasm and purified virions. In collaboration with researchers of the NCI ACVP, we have completed a structural analysis of the 1200-nt PAN in each of these cellular compartment, a goal of which was to reveal alterations on PAN occupancy by viral and/or cellular proteins. This study, recently published in Nucleic Acids Research, was extended to map the sites of recombinant KSHV proteins on in vitro transcribed PAN RNA.