Small molecules inhibitors of neuraminidases (NAs) are ones of the most prospective molecules proposed for the treatment of influenza viruses. The determination of their inhibition activity in vitro is an important step during the development of antiviral drugs. However, the analytical methods typically used for the evaluation of NA activity and inhibition (fluorescence-based assays using MUNANA substrate or thiobarbituric acid assay, TBA) may suffer from interferences caused by tested inhibitors as signal quenching or self-fluorescence, moreover in TBA are used toxic and carcinogenic reagents. The determination of the NA activity can be effectively performed by alternative methods based on lectin – glycan recognition, usually as enzyme-linked lectin assay (ELLA). We have adapted the ELLA assay to a lectin-based assay in a microplate format with fluorescence detection for determination of NA inhibitory activity. We optimized our protocol and the developed method was tested using four different small molecule NA inhibitors or potential NA inhibitors (DANA, zanamivir, quercetin and α-mangostin) with three bacterial NAs (from Clostridium perfringens, Vibrio cholerae and Arthrobacter ureafaciens), and the IC₅₀ values for NA inhibitors were determined. The inhibition effect of DANA was observed for all 3 tested NAs (IC₅₀ = 10.1 µM for V. cholerae, 13.4 µM for C. perfringens and 402.9 µM for A. ureafaciens, respectively) and of Zanamivir only for NA from V. cholerae (IC₅₀ = 101.9 µM). For both quercetin and α-mangostin, no inhibition effect to the tested NAs was observed. The main advantages of herein described method are good sensitivity due to fluorescent signal detection, the absence of the interference caused by fluorescent signal quenching by tested inhibitors, the use of natural substrates (glycoproteins) and the avoiding the use of toxic reagents.

神经氨酸酶 (NA) 的小分子抑制剂是最有前景的用于治疗流感病毒的分子之一。体外抑制活性的测定是抗病毒药物研发过程中的重要一步。然而，通常用于评估 NA 活性和抑制的分析方法（使用 MUNANA 底物的基于荧光的测定或硫代巴比妥酸测定，TBA）可能会受到测试抑制剂的干扰，如信号淬灭或自发荧光，此外在 TBA 中是使用有毒和致癌试剂。NA 活性的测定可以通过基于凝集素 - 聚糖识别的替代方法有效地进行，通常作为酶联凝集素测定 (ELLA)。我们已将 ELLA 检测调整为微孔板形式的基于凝集素的检测，并带有荧光检测，用于确定 NA 抑制活性。产气荚膜梭菌、霍乱弧菌和尿素节杆菌），并测定了 NA 抑制剂的IC ₅₀值。观察到所有3倍测试的NA DANA的抑制作用（IC ₅₀  = 10.1μM为霍乱弧菌，13.4μM为产气荚膜梭菌和402.9μM为A. ureafaciens，分别地）和扎那米韦的仅为NA从霍乱弧菌（集成电路₅₀ = 101.9 µM）。对于槲皮素和 α-芒果素，均未观察到对测试 NA 的抑制作用。本文所述方法的主要优点是由于荧光信号检测而具有良好的灵敏度、不存在由测试抑制剂引起的荧光信号猝灭引起的干扰、天然底物（糖蛋白）的使用以及避免使用有毒试剂。