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已成为全球严重的健康威胁，但新释放的抗生素数量仍然很少。解决此问题的一种方法是靶向抗性蛋白以恢复抗生素的有效性。 β-内酰胺是最成功的抗生素药物类别，但它们很容易被越来越多的β-内酰胺酶灭活。长期以来，医学上相关的β-内酰胺酶仅包含丝氨酸β-内酰胺酶（SBL），现在已经开发了几种抑制剂。然而，最近的金属β-内酰胺酶（MBL）成为一种全球威胁，尚未开发抑制剂。但是，它们在药物衰竭中的重要性日益严重是针对目标的强大动力。在这里，我们建议使用创新的管道来开发针对两种类型的β-内酰胺酶的RNase抗性抑制剂，将体外选择（SELEX）与微流体辅助的Ultrahigh-Throgh-Throughtupt筛选和下一代测序（NGS）结合起来。实际上，尽管SELEX将允许以非常大的文库的顺序富集适体，以显示结合靶蛋白的潜力，但微流体筛选将允许通过搜索这些适体确实可以抑制酶活性来完善此预选。最后，使用NGS和生物信息学将允许立即分析整个过程以迅速识别最有希望的序列。 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金黄色葡萄球菌。到该项目结束时，我们不仅将通过实际开发有效的适应症来验证这项新技术，而且我们还将开发出新的药物原型，后来可以进行优化并进入抗击细菌感染所需的阿森纳，以预见到，预见的是，在上升的数十年中，所有其他疾病都超越了所有其他疾病。此外，如本提案中讨论的那样，此处使用的DIRA管道的潜在应用谱将比抗生素发现宽得多。