One-bead-one-inhibitor-one-substrate screening of neuraminidase activity
One-bead-one-inhibitor-one-substrate screening of neuraminidase activity
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DOI：
10.1002/cbic.200500006
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发表时间：
2005-10-01
期刊：
影响因子：
3.2
通讯作者：
Gervay-Hague, J
中科院分区：
文献类型：
--
作者：
Ying, LQ;Gervay-Hague, J
鉴于21世纪世纪流感大流行的突出威胁，寻找新的抗病毒化合物是一个越来越重要的研究领域。抗病毒研究的最新进展霍韦确立了靶向病毒神经氨酸酶（NA）的可行性，该酶从细胞表面切割唾液酸，介导病毒在呼吸道中的通过。N-乙酰神经氨酸（NeuAc）是NA的底物，这种核心结构的类似物已被商业化作为抗病毒治疗剂。最近的研究已经确定，该系统非常适合用于药物发现的组合方法。这一过程中的一个重要步骤是发展固相筛选技术。本文报道了进行竞争性固相NA测定的可行性。最初，将荧光NeuAc底物固定在固体支持物上，三种NA（产气荚膜梭菌、鼠伤寒沙门氏菌和霍乱弧菌）切割底物的能力被证明与液相测定类似。然后用荧光NeuAc底物和两种已知抑制剂（DANA和扎那米韦）之一来双官能化固体支持物。NA从固体支持物上切割NeuAc的能力，同时与抑制剂一起存在时，显示为酶依赖性的。正如预期的那样，NeuAc和DANA（一种非特异性抑制剂）的同时呈现导致所有三种测试酶的活性降低。相比之下，NeuAc和选择性抑制剂扎那米韦的双重呈递仅显示出对霍乱弧菌的显著活性。
Given the eminent threat of a 21st century flu pandemic, the search for novel antiviral compounds is an increasingly important area of research. Recent developments in antiviral research hove established the viability of targeting viral neuraminidase (NA), on enzyme that cleaves sialic acid from the cell-surface-mediating passage of the virus in the respiratory tract. N-acetyl neuraminic acid (NeuAc) is the substrate for NA, and analogues of this core structure have been commercialized as antiviral therapeutics. Recent studies have established that this system is well suited for combinatorial approaches to drug discovery. An important step in the process is to develop solid-phase screening technologies. The feasibility of performing competitive solid-phase NA assays is reported herein. Initially, a fluorogenic NeuAc substrate was immobilized on solid support, and the ability of three NAs (Clostridium perfringens, Salmonella typhimurium, and Vibrio cholerae) to cleave the substrate was shown to be analogous to solution-phase assays. The solid support was then bifunctionalized with the fluorogenic NeuAc substrate and one of two known inhibitors (DANA and Zanamivir). The ability of NA to cleave NeuAc from the solid support when simultaneously presented with an inhibitor was shown to be enzyme dependent. As expected, simultaneous presentation of NeuAc and DANA, a nonspecific inhibitor, led to diminished activity for all three enzymes tested. In contrast, dual presentation of NeuAc and the selective inhibitor Zanamivir only showed significant activity against Vibrio cholerae.