Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Gilead Sciences and IOCB Research Center, Flemingovo n. 2, 16610 Prague 6, Czech Republic
Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Gilead Sciences and IOCB Research Center, Flemingovo n. 2, 16610 Prague 6, Czech Republic.
Biochem J. 2018 Dec 10;475(23):3847-3860. doi: 10.1042/BCJ20180764.
Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.
流感神经氨酸酶负责将新的病毒颗粒从感染细胞表面释放出来。临床上有几种神经氨酸酶抑制剂被用于治疗患者或储备用于紧急情况。然而，病毒对已批准的抑制剂的耐药性不断发展，突显了开发针对耐药流感株有效的新抗病毒药物的必要性。一种简便、灵敏和廉价的筛选方法将有助于实现这一目标。最近，我们描述了一种基于多孔板的 DNA 连接抑制剂抗体检测法（DIANA）。这种高度灵敏的方法可以定量检测皮摩尔浓度的酶。DIANA 还被应用于高通量酶抑制剂筛选，允许从单个抑制剂浓度评估抑制常数。在这里，我们报告了一种与报告 DNA 寡核苷酸连接的他米膦衍生物的设计、合成和结构表征，用于开发用于筛选潜在流感神经氨酸酶抑制剂的 DIANA 型测定法。神经氨酸酶首先被固定化抗体捕获，测试化合物与寡核苷酸连接的检测探针竞争与酶结合，然后通过 qPCR 定量。我们通过将其与标准荧光测定法进行比较验证了这种新的测定法，并证明了其用于灵敏神经氨酸酶检测以及潜在新神经氨酸酶抑制剂高通量筛选的有用性。
