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20世纪40年代前的情景。抗生素耐药性是全球范围内一个日益严重的问题，极大地阻碍了我们遏制结核病和疟疾等全球流行病以及简单的疟疾感染的能力。拟议的项目意义重大，对公共卫生具有巨大的影响潜力，因为除非制定创新战略来生产强大有效的新型抗生素，否则医疗保健成本将继续攀升，我们将完全失去对抗最常见感染的能力。目前的抗生素治疗主要来自真菌和细菌产生的天然产物，这些天然产物能够抑制其他生物体的生长，通常通过抑制细胞壁合成或维持或通过抑制蛋白质合成。自从弗莱明于1929年首次分离出青霉素以来，大多数后续几代抗生素仍然与最初的天然产品非常相似，其功能基团经过修饰，以增加其对更广泛病原体的活性并减少其副作用。恶唑烷酮、糖肽类、β-内酰胺类和喹诺酮类药物显示出一些前景，但革兰氏阴性菌感染仍然存在问题。核酸是药物设计的有希望的途径，既作为治疗剂又作为靶点。然而，特异性通常是小分子核酸结合剂如嵌入剂、沟结合剂和甚至氨基糖苷类的问题。在这里，我们提出了一个创新的计划，用于识别，功能和机制分析，一类新的氨基糖苷类-核酸缀合物配体，是特定的氨基糖苷类靶向核糖开关，并使其在体内失活。这种核糖开关是负责赋予数十种致病细菌菌株抗生素耐药性的机制中的关键开关，据我们所知，以前从未被靶向用于可能的治疗开发。所设计的配体是氨基糖苷缀合物，其具有对该核糖开关靶标特异性的潜力，并且可用于对抗 包括革兰氏阴性菌株在内的广谱感染性细菌。首先，如特定目标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用FRET供体和受体染料商业合成。我们将进行荧光测定，以快速筛选NUBAD LLC开发的约80种新型氨基糖苷类-核酸缀合物，用于结合核糖开关靶标，并鉴定对靶标核糖开关具有高特异性和亲和力的有前途的配体（如特定目标2所述）。将使用体内试验（特定目标2）来鉴定被氨基糖苷类耐药细胞摄取并使其再次对氨基糖苷类敏感的先导化合物。为了验证化合物确实抑制核糖开关的作用机制，将进行机制测定（具体目标3）。核糖开关将位于报告质粒内，使得其处于IPTG诱导型tac启动子（Ptac）的控制下，所述启动子将位于β-gal报告基因的上游。在存在氨基糖苷类和选定的已鉴定缀合配体结合剂的情况下，通过琼脂扩散分析评估核糖开关的功能。作为本研究的结果，将鉴定出几种先导化合物：（1）被病原菌吸收;（2）恢复氨基糖苷类药物对耐药菌的敏感性;（3）特异性靶向氨基糖苷类药物结合核糖开关作为其主要作用机制。该项目的未来阶段将重点开发这些先导化合物作为治疗药物。NUBAD LLC是一家药物发现公司，致力于识别靶向核酸的治疗剂。我们开发针对RNA和DNA结构的新型探针、测定和小分子疗法，这些结构被确定为人类疾病的靶点，该项目非常适合NUBAD的目标及其员工的特定技能。