A biophysical assay targeting Gyrase RNA

项目摘要 在抗击传染病方面，世界正迅速走向1940年前的局面。 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 世界范围的流行病，如结核病和疟疾，以及简单的疟疾感染。 拟议的项目意义重大，因为除非制定创新战略， 生产强大而有效的新型抗生素，医疗保健成本将继续攀升 我们将完全丧失抵抗最常见传染病的能力。电流 抗生素治疗主要来源于真菌和细菌产生的天然产物， 能够抑制其他生物的生长，通常是通过抑制细胞壁的合成或维持 或通过抑制蛋白质合成。自从1929年弗莱明首次分离出青霉素以来， 随后的几代抗生素仍然非常类似于原始的天然产物， 修改组，以增加其对更广泛的病原体的活性，并减少其侧 影响剖面。恶唑烷酮、糖肽、β-内酰胺和喹诺酮类药物显示出一些前景， 未来，但革兰氏阴性细菌感染仍然存在问题。 多药耐药（MDR，耐2 - 3类）、广泛耐药（XDR，耐 大多数类别，除了粘菌素或替加环素），甚至泛耐药（PDR，耐所有类别） 近年来，医院内细菌感染激增， 分离株使这些感染越来越难以治疗。像这样的医院获得性感染 占美国所有住院人数的4%，并且致病病原体的多样性令人难以置信， 抗生素耐药性和严重程度。医院感染的一个重要原因是肠杆菌科 革兰氏阴性杆菌科，包括革兰氏阴性杆菌，可以是细菌性或致病性的。肠杆菌科有 由于它们引起的感染的多样性，造成广泛的临床和经济影响;该家族引起 许多感染，如肺炎、血流感染（BSI）、尿路感染（UTI）和肠内感染。 腹腔感染（IAI）。世界卫生组织（WHO）将耐碳青霉烯类药物 肠杆菌科（CRE）作为其优先病原体列表中对新型抗生素的迫切需求。 因为这些多重耐药感染的死亡率在30%到50%之间， 在寻找可行的治疗方法时，必须解决对这些病原体的新疗法的需求。 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。重点瞄准 保守的RNA序列和结构，在细菌（肠杆菌科），并参与增殖和 细菌的存活，是一种很有前途的方法。使用我们专有的探针、分析和文库， 建议开发一种筛选肠杆菌科中必需基因的方法。在这里，我们提出一个 用于鉴定一类新配体的创新计划，所述配体对存在于 细菌中的一个必需基因，我们提出了一种生物物理筛选试验，用于识别这种基因， 配体。首先，如在具体目标1中所概述的，我们将表征将被分析的模型核酸结构域。 商业合成并鉴定特异性和高亲和力氨基糖结合剂。然后我们将合成 序列特异性RNA结合配体，并筛选该靶向缀合物文库的序列特异性 结合和基因抑制。作用机制将使用报告基因测定法（特异性 目标2）。该方法的成功应用将使我们能够识别和验证先导化合物 在II期研究中抑制细菌生长。

PROJECT SUMMARY 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 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 penicillin was first isolated by Fleming in 1929, most of the subsequent generations of antibiotics remain very similar to the original natural products, wit h 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. Cases of multidrug-resistant (MDR, resistance to 2-3 classes), extensive drug resistance (XDR, resistance to most classes except colistin or tigecycline) and even pan drug resistance (PDR, resistance to all classes) nosocomial bacterial infections have skyrocketed in recent years, and the emergence of pan drug-resistant isolates are making these infections increasingly difficult to treat. Hospital-acquired infections like these account for up to 4% of all hospital stays in the United States and are incredibly diverse in causative pathogen, antibiotic resistance profile, and severity. A significant cause of nosocomial infection is the Enterobacteriaceae family, which includes Gram-negative bacilli that can be commensal or pathogenic. Enterobacteriaceae have a widespread clinical and economic impact due to the diversity of infections they cause; this family causes many infections such as pneumonia, bloodstream infections (BSIs), urinary tract infections (UTIs), and intra- abdominal infections (IAIs). The World Health Organization (WHO) lists carbapenem-resistant Enterobacteriaceae (CRE) as having a critical need for novel antibiotics on their Priority Pathogens list. Because the mortality of these multi drug-resistant infections is between 30 and 50% and there is such difficulty in finding viable treatments, the need for novel therapeutics for these pathogens must be addressed. Nucleic acids are promising avenues for drug design, both as therapeutics and as targets. Targeting heavily conserved RNA sequences and structures, in bacteria (Enterobacteriaceae), and involved in proliferation and survival of bacteria, is a promising approach. Using our proprietary probes, assays and libraries, we propose to develop a screening assay for an essential gene in Enterobacteriaceae. Here we propose an innovative plan for identification of a novel class of ligands that are specific for an mRNA present in an essential gene in bacteria, and we propose a biophysical screening assay for identifying such ligands. First, as outlined in Specific Aim 1, we will characterize a model nucleic acid domain that will be synthesized commercially and identify specific and high-affinity aminosugar binders. We will then synthesize sequence-specific RNA binding ligands and screen this targeted library of conjugates for sequence-specific binding and gene inhibition. The mechanism of action will be confirmed using a reporter gene assay (Specific Aim 2). A successful application of the approach will allow us to identify and validate lead compounds for inhibition of bacterial growth in Phase II studies.