Development of Aminoglycoside-Nucleic Acid Conjugates for Inactivation of an Antibiotic Resistance-Conferring Aminoglycoside Sensing Riboswitch

氨基糖苷-核酸缀合物的开发用于灭活赋予抗生素抗性的氨基糖苷传感核糖开关

• 批准号: 9015742
• 负责人: sandra Paige story
• 金额: $ 21.2万
• 依托单位: NUBAD, LLC
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9015742
• 关键词: Adverse effects; Affinity; Agar; Aminoglycoside Antibiotics; Aminoglycoside resistance; Aminoglycosides; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antimicrobial Resistance; Antimicrobial susceptibility; Bacteria; Bacterial Infections; Bacterial RNA; Binding; Biological Assay; Boston; Cell Line; Cell Wall; Cells; Communicable Diseases; DNA Structure; Development; Diffusion; Drug Design; Drug Targeting; Drug resistance; Dyes; Elements; Employee; Enzymes; Epidemic; Fluorescence; Fluorescence Resonance Energy Transfer; Future; Generations; Genetic; Glycopeptides; Goals; Gram-Negative Bacterial Infections; Growth; Head; Health; Health Care Costs; Housing; In Vitro; Infection; Institute of Medicine (U.S.); Intercalating Agents; Isopropyl Thiogalactoside; Label; Lactams; Lead; Ligand Binding; Ligands; Maintenance; Malaria; Marketing; Modeling; Natural Products; Nosocomial Infections; Nucleic Acids; Organism; Parasites; Pathway interactions; Pharmaceutical Preparations; Phase; Plasmids; Positioning Attribute; Predisposition; Protein Biosynthesis; Public Health; Quinolones; RNA; RNA Interference; Reader; Reporter; Reporter Genes; Research; Scanning; Specificity; Staphylococcal Infections; Testing; Therapeutic; Transferase; Tuberculosis; United States; United States National Academy of Sciences; Virus; Work; analog; antimicrobial; antimicrobial drug; aptamer; bacterial resistance; combat; cost; design; dosage; drug discovery; drug modification; fighting; fluorophore; functional group; fungus; human disease; in vivo; innovation; microorganism; novel; pathogen; pathogenic bacteria; prevent; promoter; research study; resistance gene; restoration; skills; small molecule; small molecule therapeutics; targeted treatment; therapeutic development; therapeutic target; uptake

 DESCRIPTION (provided by applicant): The world is rapidly heading towards a pre-1940's scenario when it comes to fighting infectious disease. Antimicrobial resistance is a growing problem on a global scale, greatly hampering our abilities to quell worldwide epidemics such as tuberculosis and malaria, as well as the simple staphylococcus infection. The proposed project is significant and has huge potential for impact on public health because unless innovative strategies are developed to produce robust and effective new classes of antibiotics, health care costs will continue to climb and we will completely lose our ability to combat even the most common infection. Current antibiotic treatments originated predominantly from natural products produced by fungi and bacteria that were able to inhibit the growth of other organisms, usually by inhibiting cell wall synthesis or maintenance or by inhibiting protein synthesis. Since penicilln was first isolated by Fleming in 1929, most of the subsequent generations of antibiotics remain very similar to the original natural products, with functional groups modified to increase their activity across a broader range of pathogens and decrease their side effect profiles. Oxazolidones, glycopeptides, -lactams, and quinolones show some promise for the future, but gram-negative bacterial infections still remain problematic. Nucleic acids are promising avenues for drug design, both as therapeutics and as targets. However, specificity is often a problem with small molecule nucleic acid binders such as intercalators, groove binders, and even aminoglycosides. Here we propose an innovative plan for identification of, and both functional and mechanistic assaying of, a novel class of aminoglycoside-nucleic acid conjugate ligands that are specific for an aminoglycoside-targeting riboswitch and render it inactive in vivo. This riboswitch is a key switch in the mechanism responsible for conferring antibiotic resistance in dozens of pathogenic bacterial strains, and has never before been targeted for possible therapeutic development, to our knowledge. The designed ligands, which are aminoglycoside conjugates, have the potential to be both specific for this riboswitch target, and useful against a broad spectrum of infectious bacteria, including gram- negative strains. First, as outlined in Specific Aim 1, we will obtain a model riboswitch aptamer domain that has been synthesized commercially with FRET donor and acceptor dyes in different regions of the construct. We will perform a fluorescence assay to rapidly screen approximately 80 novel aminoglycoside-nucleic acid conjugates developed at NUBAD LLC for binding to the riboswitch target, and identify promising ligands with high specificity and affinity for the target riboswitch (as outlined in Specific Aim 2). In vivo assays will be used (Specific Aim 2) to identify lead compounds that are uptaken by aminoglycoside resistant cells and render them susceptible aminoglycosides once again. In order to verify that the compounds indeed inhibit the riboswitch's mechanism of action, mechanistic assays will be performed (Specific Aim 3). The riboswitch will be positioned within a reporter plasmid so that it is under control of an IPTG-inducible tac promoter (Ptac) that will be positioned upstream of the -gal reporter gene. Function of the riboswitch will be assessed by agar diffusion analysis in the presence of aminoglycosides and selected identified conjugate ligand binders. As a result of this study, several lead compounds will be identified that (1) are taken up by pathogenic bacteria; (2) restore aminoglycoside susceptibility to resistant bacteria, and (3) specifically target the aminoglycoside-binding riboswitch as their primary mechanism of action. Future phases of this project will focus on developing these lead compounds for development as therapeutics. NUBAD LLC is a drug discovery company devoted to identifying therapeutic agents that target nucleic acids. We develop novel probes, assays and small molecule therapeutics targeting RNA and DNA structures identified as targets in human disease, and this project is extremely well-suited to NUBAD's aims and its employees' specific skill sets.

 描述（由申请人提供）：在抗击传染病方面，世界正迅速走向 1940 年代之前的局面。抗生素耐药性是全球范围内日益严重的问题，极大地阻碍了我们平息结核病和疟疾等全球流行病的能力。以及简单的葡萄球菌感染，拟议的项目意义重大，对公众健康具有巨大的影响潜力，因为除非开发出强有力的、有效的新型抗生素的创新策略，否则健康将受到影响。医疗费用将继续攀升，我们将完全失去抵抗最常见感染的能力。目前的抗生素治疗主要源自真菌和细菌产生的天然产物，这些天然产物通常通过抑制细胞壁来抑制其他生物体的生长。自 1929 年弗莱明首次分离出青霉素以来，大多数后续世代的抗生素仍然与原始天然产物非常相似，其功能基团经过修饰，以提高其对更广泛病原体的活性。减少它们的副作用恶唑烷酮、糖肽、β-内酰胺和喹诺酮类药物在未来具有一定的前景，但革兰氏阴性细菌感染仍然是药物设计的有前途的途径，无论是作为治疗还是作为靶点。在此，我们提出了一种创新计划，用于识别以及功能和机械分析，例如嵌入剂、凹槽结合剂、甚至氨基糖苷类小分子核酸结合剂。一类新型氨基糖苷-核酸缀合物配体，对氨基糖苷靶向核糖开关具有特异性，并使其在体内失活。这种核糖开关是导致数十种致病细菌菌株产生抗生素耐药性的机制中的关键开关，并且从未出现过这种情况。据我们所知，设计的配体（氨基糖苷缀合物）在被用于可能的治疗开发之前，有可能对该核糖开关靶标具有特异性，并且可用于对抗核糖开关靶点。 首先，如具体目标 1 中所述，我们将获得一个模型核糖开关适体结构域，该结构域已在构建体的不同区域中使用 FRET 供体和受体染料进行商业合成。一种荧光测定法，可快速筛选 NUBAD LLC 开发的约 80 种新型氨基糖苷-核酸缀合物，用于与核糖开关靶标结合，并识别对靶标具有高特异性和亲和力的有前途的配体核糖开关（如具体目标 2 中所述）将用于鉴定被氨基糖苷类耐药细胞摄取的先导化合物，并使其再次对氨基糖苷类敏感，以验证这些化合物确实抑制。核糖开关的作用机制，将进行机械分析（具体目标 3） 核糖开关将被定位在报告质粒内，以便它处于报告基因的控制之下。 IPTG 诱导的 tac 启动子 (Ptac) 位于 -gal 报告基因的上游，将在氨基糖苷类和选定的缀合配体结合物存在的情况下通过琼脂扩散分析进行评估。 ，将鉴定出几种先导化合物：（1）被病原菌吸收；（2）恢复对耐药细菌的氨基糖苷敏感性，以及（3）特异性靶向氨基糖苷结合。 NUBAD LLC 是一家致力于识别靶向核酸的治疗药物的公司，该项目的未来阶段将重点开发这些先导化合物。针对被确定为人类疾病目标的 RNA 和 DNA 结构的分子疗法，该项目非常适合 NUBAD 的目标及其员工的特定技能。