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严重的健康威胁，但新发布的抗生素数量仍然很低。解决这一问题的一种方法是靶向耐药蛋白以恢复抗生素的功效。β-内酰胺类是最成功的一类抗生素药物，但它们容易被越来越多的β-内酰胺酶灭活。长期以来，医学上相关的β-内酰胺酶仅包括丝氨酸β-内酰胺酶（SBL），现在已经开发了几种抑制剂。然而，最近金属-β-内酰胺酶（MBL）成为全球性的威胁，尚未开发出抑制剂。然而，它们在药物失败中的重要性日益增加，这是针对它们的强烈动机。在这里，我们建议使用一种创新的管道，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）相结合，开发针对两种类型β-内酰胺酶的RNA酶抗性抑制性适体。事实上，尽管SELEX将允许从显示结合靶蛋白的潜力的序列中的非常大的文库中富集适体，但微流体筛选将允许通过搜索真正能够抑制酶活性的那些适体来细化这种预选。最后，使用NGS和生物信息学将允许一次分析整个过程，以快速识别最有希望的序列。该管道的大多数关键元素已经在初步实验中得到验证，使该项目的大部分重点放在能够抑制三种医学相关胞外酶的新适体的实际开发上：来自肺炎克雷伯氏菌的金属-β-内酰胺酶NDM-1和丝氨酸-β-内酰胺酶BlaZ和蛋白酶Aureolysin，两者都来自金黄色葡萄球菌。在这个项目结束时，我们不仅将通过有效适体的实际开发来验证这项新技术，而且我们还将开发新的药物原型，这些药物可以在以后进行优化并进入对抗细菌感染所需的武器库，预计这将是未来几十年内超越其他疾病的主要线索。此外，DIRA管道的潜在应用范围将比本提案中讨论的抗生素发现广泛得多。