Wang Zi-Qiang, Xuan Teng-Fei, Liu Jun, Chen Wei-Min, Lin Jing
College of Pharmacy, Jinan University Guangzhou 510632 P. R. China
RSC Adv. 2020 May 21;10(33):19482-19489. doi: 10.1039/d0ra02540b. eCollection 2020 May 20.
The dinucleotide 3',5'-cyclic diguanylic acid (c-di-GMP) is a critical second messenger found in bacteria. High cellular levels of c-di-GMP are associated with a sessile, biofilm lifestyle in many bacteria, which is associated with more than 70% of clinically resistant infections. Cellular c-di-GMP concentrations are regulated by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), which are responsible for the production and degradation, respectively, of c-di-GMP. Therefore, DGCs and PDEs might be attractive drug targets for controlling biofilm formation. In this study, a simple and universal high-throughput method based on a c-di-GMP-specific fluorescent probe for the determination of DGC and PDE activity was described. By using the proposed method, the c-di-GMP content in samples was rapidly quantified by measuring the fluorescence intensity in a 96-well plate by using a microplate reader. In addition, the probe molecule A18 directly interacted with the substrate c-di-GMP, and the method was not limited by the structure of enzymes.
二核苷酸3',5'-环二鸟苷酸（c-di-GMP）是一种在细菌中发现的关键第二信使。在许多细菌中，细胞内高水平的c-di-GMP与固着的生物膜生活方式相关，而这种生活方式与超过70%的临床耐药感染有关。细胞内c-di-GMP的浓度由二鸟苷酸环化酶（DGCs）和磷酸二酯酶（PDEs）调节，它们分别负责c-di-GMP的产生和降解。因此，DGCs和PDEs可能是控制生物膜形成的有吸引力的药物靶点。在本研究中，描述了一种基于c-di-GMP特异性荧光探针的简单通用的高通量方法，用于测定DGC和PDE的活性。通过使用所提出的方法，通过使用微孔板读数器测量96孔板中的荧光强度，快速定量样品中的c-di-GMP含量。此外，探针分子A18直接与底物c-di-GMP相互作用，该方法不受酶结构的限制。
