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

 描述(申请人提供)：在抗击传染病方面，世界正迅速走向1940年前的S情景。抗菌素耐药性在全球范围内是一个日益严重的问题，极大地阻碍了我们平息结核病和疟疾等全球流行病以及简单的葡萄球菌感染的能力。拟议的项目意义重大，对公共卫生有巨大的潜在影响，因为除非开发出创新的战略来生产强大和有效的新类别抗生素，否则医疗保健成本将继续攀升，我们将完全失去抗击最常见感染的能力。目前的抗生素治疗主要来自真菌和细菌产生的天然产品，这些产品能够抑制其他生物的生长，通常通过抑制细胞壁合成或维护或通过抑制蛋白质合成来实现。自从1929年弗莱明首次分离出青霉素以来，后来的大多数抗生素都与最初的天然产品非常相似，功能基团经过修改后，可以提高它们对更广泛病原体的活性，并减少它们的副作用。恶唑烷酮类、糖肽类、-内酰胺类和喹诺酮类药物显示了一些未来的希望，但革兰氏阴性细菌感染仍然存在问题。核酸是药物设计的很有前途的途径，既是治疗药物，也是靶点。然而，特异性往往是小分子核酸结合剂的问题，如嵌入剂、沟槽结合剂，甚至氨基糖苷类。在这里，我们提出了一个创新的计划，用于鉴定一类新型的氨基糖苷类核酸共轭配体，并对其进行功能和机制分析，这些配体是针对氨基糖苷类靶向核糖开关的，并使其在体内失去活性。据我们所知，这种核糖开关是导致数十种致病细菌产生抗生素耐药性的机制的关键开关，以前从未成为可能的治疗开发的目标。设计的配体是氨基糖苷类结合物，有可能既是这种核糖开关靶标的特异性配体，又可以有效地对抗 广泛的感染性细菌，包括革兰氏阴性菌株。首先，如特定目标1所述，我们将获得一个模型核糖开关适配子结构域，该结构域已用FRET供体和受体染料在构建的不同区域进行商业合成。我们将进行荧光分析，以快速筛选NUBAD LLC开发的大约80种与核糖开关靶标结合的新型氨基糖苷核酸结合物，并确定与目标核糖开关具有高特异性和亲和力的有希望的配体(如特定目标2中所概述的)。体内试验将被使用(特定目标2)来识别被氨基糖苷耐药细胞摄取的先导化合物，并使它们再次对氨基糖苷类药物敏感。为了证实这些化合物确实抑制了核糖开关的作用机制，将进行机械分析(具体目标3)。该核糖开关将定位在报告质粒内，从而受位于-GAL报告基因上游的iptg诱导的tac启动子(Pac)的控制。核糖开关的功能将通过琼脂扩散分析在氨基糖苷类和选定的识别的共轭配体结合的存在下进行评估。作为这项研究的结果，将确定几种先导化合物：(1)被病原菌摄取；(2)恢复氨基糖苷对耐药细菌的敏感性；(3)将氨基糖苷结合核糖开关作为其主要作用机制。该项目的未来阶段将侧重于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，致力于识别针对核酸的治疗剂。我们针对被确认为人类疾病靶点的RNA和DNA结构开发新的探针、分析和小分子疗法，该项目非常适合NUBAD的目标及其员工的特定技能集。

 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.