Development of inhibitory RNA aptamers using ultrahigh-throughput screening to fight antibiotic resistance.

使用超高通量筛选开发抑制性 RNA 适体来对抗抗生素耐药性。

• 批准号: 431327544
• 负责人: Professorin Dr. Beatrix Süß
• 金额: --
• 依托单位: Fachgebiet Molecular Genetics
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/431327544?language=en
• 关键词: Development; inhibitory; RNA; aptamers; using

The increase of antibiotic resistance is becoming a serious health threat worldwide, but the number of newly released antibiotics remains low. One way to address this problem is to target resistance proteins to restore the efficacy of antibiotics. β-lactams are the most successful class of antibiotic drugs but they are vulnerable to inactivation by a growing class of β-lactamases. For a long time, medically relevant β-lactamases encompassed only serine β-lactamases (SBLs) for which several inhibitors have now been developed. However, more recently metallo-β-lactamases (MBLs) emerged as a global threat for which no inhibitor has been developed yet. However, their increasing importance in drug failure is a strong incentive to target them. Here, we propose to develop RNase resistant inhibitory aptamers targeting both types of β-lactamases using an innovative pipeline combining the use of in vitro selection (SELEX) in tandem with microfluidic-assisted ultrahigh-throughput screening and next generation sequencing (NGS). Indeed, whereas SELEX will allow to enrich aptamers from very large libraries in sequence displaying the potential to bind the target protein, microfluidic screening will allow refining this pre-selection by searching for those aptamers really able to inhibit enzyme activity. Finally, using NGS and bioinformatics will allow analyzing the whole process at once to rapidly identify most promising sequences. Most key elements of this pipeline have already been validated during preliminary experiments, allowing the majority of the project to be focused on the actual development of new aptamers able to inhibit three medically relevant extracellular enzymes: the metallo-β-lactamase NDM-1 from Klebsiella pneumoniae and the serine-β-lactamases BlaZ and the protease Aureolysin, both from Staphylococcus aureus. By the end of this project we will not only have validated this new technology through the actual development of efficient aptamers, but we will also have developed new prototypes of drugs that could later be optimized and enter into the arsenal required to fight bacterial infection that are foreseen to be a major thread surpassing every other disease in the up-coming decades. Moreover, the potential application spectrum of the DIRA pipeline refined and used here will be much wider than antibiotic discovery as discussed in the present proposal.

抗生素耐药性的增加正在成为全球范围内的严重健康威胁，但解决这一问题的一种方法是针对耐药蛋白来恢复β-内酰胺类抗生素的功效。抗生素药物，但它们很容易被越来越多的 β-内酰胺酶失活 长期以来，医学相关的 β-内酰胺酶仅包括丝氨酸 β-内酰胺酶 (SBL)，现已开发出几种抑制剂。然而，最近，金属-β-内酰胺酶 (MBL) 成为一种全球性威胁，目前尚未开发出相应的抑制剂。然而，它们在药物失败中的重要性日益增加，因此我们建议开发 RNase 抗性药物。使用创新的管道将体外选择 (SELEX) 与微流体辅助超高通量筛选和下一代测序 (NGS) 结合起来，针对两种类型的 β-内酰胺酶抑制性适体。 SELEX 可以从非常大的文库中按序列富集显示出与靶蛋白结合潜力的适体，而微流体筛选将通过搜索那些真正能够抑制酶活性的适体来改进这种预选。最后，使用 NGS 和生物信息学将允许。允许立即分析整个过程，以快速识别最有希望的序列。该管道的大多数关键要素已在初步实验中得到验证，从而使该项目的大部分内容可以集中于新适体的实际开发。能够抑制三种医学相关的细胞外酶：来自肺炎克雷伯菌的金属-β-内酰胺酶 NDM-1 和来自金黄色葡萄球菌的丝氨酸-β-内酰胺酶 BlaZ 和蛋白酶金黄色素溶血素。通过高效适体的实际开发验证了这项新技术，但我们还将开发出新的药物原型，这些药物原型可以在以后进行优化并进入临床试验。预计细菌感染将成为未来几十年超越所有其他疾病的主要途径。此外，这里改进和使用的 DIRA 管道的潜在应用范围将比抗生素的发现广泛得多。提出建议。