Nucleic Acid Footprinting and development of small molecule antagonists

核酸足迹和小分子拮抗剂的开发

• 批准号: 9556297
• 负责人: Stuart F. J. Le Grice
• 金额: $ 77.74万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9556297
• 关键词: Address; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Biological Process; Biological Testing; Cell Nucleus; Chemicals; Collaborations; Collection; Comorbidity; Complex; Coupled; Cytoplasm; Dengue Virus; Development; Dimerization; Disease; Drug Design; Elements; Enzymes; Etiology; Future; Generations; Genetic Transcription; Genome; Goals; HIV; HIV Genome; HIV-1; Hepatitis B; Herpesviridae; In Vitro; Investigation; Kaposi Sarcoma; Label; Laboratories; Latent Virus; Lead; Length; Libraries; Ligands; Literature; Maps; Masks; Mediating; Methods; Microscope; Modification; Molecular; Molecular Conformation; Molecular Target; Multicentric Angiofollicular Lymphoid Hyperplasia; NMR Spectroscopy; Natural Products; Nature; Nuclear; Nuclear RNA; Nucleic Acids; Nucleotides; Pharmaceutical Chemistry; Pilot Projects; Poly A; Post-Transcriptional RNA Processing; Primary carcinoma of the liver cells; Process; Production; Proteins; Publishing; RNA; RNA Binding; RNA Probes; RNA Transport; Recombinants; Regulation; Reproducibility; Research; Research Personnel; Resolution; Response Elements; Ribosomal Frameshifting; Signal Transduction; Site; Slide; Structure; System; Techniques; Technology; Therapeutic; Transactivation; Untranslated RNA; Viral; Viral Genome; Viral Packaging; Viral Pathogenesis; Viral Proteins; Virion; Virus; Virus Replication; base; biophysical techniques; cost; design; dimer; genomic RNA; high throughput screening; inhibitor/antagonist; killings; liquid chromatography mass spectrometry; novel; novel strategies; novel therapeutics; nucleocytoplasmic transport; nucleotidyltransferase; pharmacophore; primary effusion lymphoma; rev Protein; screening; small molecule; small molecule libraries; stem; targeted treatment; therapeutic target; triple helix; viral RNA

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.

内部的努力导致了小分子微阵列的开发和实施，上面印有 20,000 个小分子的库。荧光标记的 RNA（长度为 60 个核苷酸）流过阵列，结合被记录为荧光信号。重要的是，整个阵列可以在一周内进行探测，并且节省 RNA 量。使用该系统，我们成功地鉴定了在生化测定中识别 HIV-1 反式激活反应 (TAR) 元件并抑制培养物中病毒复制的新化学型。药物化学成功地衍生出了新的化学型，其结合位点通过核磁共振波谱法进行了鉴定。这个“试点”项目是扩大利用小分子靶向调控 RNAS 的努力的基础。正在研究的第二个 HIV 顺式作用 RNA 是 Rev 反应元件 (RRE)，它对于病毒基因组的核细胞质运输非常重要。 RRE 的干环 IIB (SLIIB) 提供了 HIV Rev 蛋白的主要结合位点，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。我们扩展了这样的观察结果，以探究 m6A 修饰的 RNA 是否为小分子识别提供独特的信号。事实上，使用我们的小分子微阵列，除了识别这两种 RNA 的第三类之外，还鉴定出了识别非甲基化或 m6A 甲基化 RRE SLIIB 的化学型。正在了解 m6A 修饰诱导的选择性小分子识别构象变化的性质。将小分子微阵列策略应用于 KSHV PAN lncRNA 的 ENE 元件，已鉴定出识别三螺旋的化学型。生物学测试表明，这些化合物的一部分不具有细胞毒性，这使我们能够研究它们对 KSHV 复制的影响。出乎意料的是，一种化合物能够重新激活潜伏的 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，这为开发“踢杀”策略提供了可能性，通过该策略，激活的病毒可以被第二组针对特定病毒酶的小分子所靶向。正如稍后将概述的，实验室的一个平行项目确实鉴定出了针对一种或多种 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视为一种治疗策略的选择。因此，研究人员对 ENE 的双链体版本进行了研究，它揭示了额外的、独特的化学型。计划在不久的将来对这些化合物进行生物测试。该转化项目与生物化学（SHAPE-MaP）和生物物理方法（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小分子与显微镜载玻片共价连接，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解决方案的筛选策略，该策略更适合 NCI 分子靶标实验室 (MTL) 收集的大量小分子，特别是其大量的天然产物提取物。使用登革热病毒 RNA 基因组的茎环 A (SLA) 作为模型系统，我们开发了一种低成本、稳健且可重复的高通量热变性测定 (HTS-Thermoflor)，利用该方法从 MTL 中鉴定出新的化学型纯天然产品的集合。我们还表明，相同的策略可以应用于天然产物提取物的 MTL 集合，它代表了新颖化学实体的独特集合。随后将通过 LC/MS 完成 SLA 结合化学型的鉴定。 250,000个分子的全筛选需要20毫克纯化RNA，并且通过致力于调节RNA的NMR分析，该实验室拥有足够的能力进行高水平RNA生产和纯化。 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体中。我们与 NCI ACVP 的研究人员合作，完成了每个细胞区室中 1200 nt PAN 的结构分析，其目标是揭示病毒和/或细胞蛋白对 PAN 占据的变化。这项研究最近发表在《Nucleic Acids Research》上，该研究扩展到在体外转录的 PAN RNA 上绘制重组 KSHV 蛋白的位点。