Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, 06520-8103, USA.
L2 Diagnostics, LLC, 300 George Street, New Haven, CT, 06511, USA.
Anal Biochem. 2023 Apr 1;666:115047. doi: 10.1016/j.ab.2023.115047. Epub 2023 Jan 20.
Due to the emergence of multidrug resistant pathogens, it is imperative to identify new targets for antibiotic drug discovery. The S-adenosylhomocysteine (SAH) nucleosidase enzyme is a promising target for antimicrobial drug development due to its critical functions in multiple bacterial processes including recycling of toxic byproducts of S-adenosylmethionine (SAM)-mediated reactions and producing the precursor of the universal quorum sensing signal, autoinducer-2 (AI-2). Riboswitches are structured RNA elements typically used by bacteria to precisely monitor and respond to changes in essential bacterial processes, including metabolism. Natural riboswitches fused to a reporter gene can be exploited to detect changes in metabolism or in physiological signaling. We performed a high-throughput screen (HTS) using an SAH-riboswitch controlled β-galactosidase reporter gene in Escherichia coli to discover small molecules that inhibit SAH recycling. We demonstrate that the assay strategy using SAH riboswitches to detect the effects of SAH nucleosidase inhibitors can quickly identify compounds that penetrate the barriers of Gram-negative bacterial cells and perturb pathways involving SAH.
由于多药耐药病原体的出现，迫切需要确定抗生素药物发现的新靶点。S-腺苷同型半胱氨酸（SAH）核苷酶是一种有前途的抗菌药物开发靶点，因为它在多种细菌过程中具有关键功能，包括回收 S-腺苷甲硫氨酸（SAM）介导的反应产生的有毒副产物和产生通用群体感应信号前体，自诱导物-2（AI-2）。核酶是一种结构 RNA 元件，细菌通常利用它来精确监测和响应包括代谢在内的基本细菌过程的变化。与报告基因融合的天然核酶可以用来检测代谢或生理信号的变化。我们在大肠杆菌中使用 SAH 核酶控制的β-半乳糖苷酶报告基因进行了高通量筛选（HTS），以发现抑制 SAH 循环的小分子。我们证明，使用 SAH 核酶检测 SAH 核苷酶抑制剂作用的测定策略可以快速识别穿透革兰氏阴性细菌细胞屏障并扰乱涉及 SAH 的途径的化合物。
