流感病毒神经氨酸酶抑制剂筛选模型的建立和应用

目的建立适用于高通量筛选的流感病毒神经氨酸酶(neuraminidase,NA)抑制剂筛选模型。方法从甲型及乙型流感病毒中制备神经氨酸酶,以2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid(MUNANA)作为底物,建立检测神经氨酸酶活性的荧光测定法及其抑制剂体外筛选方法,用高通量筛选系统对1 200个化合物与提取物进行初筛。结果神经氨酸酶酶促反应以pH 3.5,二价阳离子浓度为2～6 mmol·L^-1及37℃孵育时酶活性最佳;甲、乙型流感病毒不同株神经氨酸酶的米氏常数(Km)的范围为(4.89～5.94) μmol·L^-1;初筛发现12个化合物对流感病毒神经氨酸酶有可重复的抑制活性。结论优化了神经氨酸酶反应体系,建立的体外模型可用于抗甲、乙型流感病毒药物的高通量筛选及酶抑制动力学的研究。

ESTABLISHMENT AND APPLICATION OF HIGH THROUGHPUT SCREENING MODEL FOR INFLUENZA VIRUS NEURAMINIDASE INHIBITORS IN VITRO

AIMIn order to find novel inhibitors of influenza virus neuraminidase (NA), an assay method of neuraminidase activity was established for high throughput screening. METHODSThe strain A (Yuefang 72-243 A and Jifang 90-15 A) and B (Sichuan 2000-38 B) influenza viruses were used as source of neuraminidase and the activity of neuraminidase was measured by fluorometric method. The reaction system of neuraminidase with substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MUNANA) was optimized by changing the conditions such as concentrations of neuraminidase, substrate, Ca²⁺, reactive system pH and temperature. At last, the inhibitory effects of 1 200 samples (including compounds and extracts from plants) were screened by the fluorometric assay. RESULTS The optimized neuraminidase reaction system contains enzyme, MUNANA 20 μmol·L^-1, Ca²⁺ 4 mmol·L^-1, at pH 3.5 and 37℃. The Michaelis and Menten constants (Km) of influenza virus A (Yuefang 72-243 and Jifang 90-15) and B (Sichuan 2000-38) are (5.9±1.9), (5.2±0.5) and (4.9±1.2) μmol·L^-1 respectively. Among 1 200 samples, there were about 1% showed potential inhibitory effects on influenza virus neuraminidase. CONCLUSION The temperature, pH and concentrations of substrate and ions are very important factors affecting the influenza virus neuraminidase activity in vitro. The fluorometric assay of neuraminidase was performed by automatic laboratory station to screen inhibitors of influenza virus neuraminidase in vitro. It can also be used to study the enzyme inhibition kinetics.