Department of System Chemotherapy and Molecular Sciences, Division of Bioinformatics and Chemical Genomics, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto, 606-8501, Japan.
Chembiochem. 2016 Mar 15;17(6):474-8. doi: 10.1002/cbic.201500553. Epub 2016 Feb 16.
We describe a proof-of-concept study of a competitive enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) with active-site-directed probes coupled to a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold. A biotin functionality immobilizes the probes onto a streptavidin-coated solid support. Dissociation constants were determined with a series of ligands, including enzyme substrates and a library of sulfamoyloxy-linked aminoacyl/aryl-AMP analogues. As it enables direct readout of protein-ligand interaction, the competitive ELISA technique provided information on comparative structure- activity relationships and insights into the enzyme active-site architecture of NRPS A-domains. These studies indicate that the ELISA technique can accelerate the discovery of small-molecule inhibitors of the A-domains with new scaffolds that perturb the production of NRPS-related virulence factors.
我们描述了一种针对非核糖体肽合成酶（NRPS）A 结构域的具有活性位点定向探针的竞争性酶联免疫吸附测定（ELISA）系统的概念验证研究，该探针与 5'-O-N-（氨酰基）磺酰腺苷支架偶联。生物素功能将探针固定在链霉亲和素包被的固体载体上。通过一系列配体（包括酶底物和磺酰胺氧基连接的氨酰/芳基-AMP 类似物文库）确定解离常数。由于它能够直接读出蛋白质-配体相互作用，竞争性 ELISA 技术提供了关于比较结构-活性关系的信息，并深入了解了 NRPS A 结构域的酶活性位点结构。这些研究表明，ELISA 技术可以加速发现具有新支架的 A 结构域的小分子抑制剂，这些支架可以干扰 NRPS 相关毒力因子的产生。
