Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
J Virol. 2010 May;84(9):4277-88. doi: 10.1128/JVI.02325-09. Epub 2010 Feb 24.
A new class of N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide inhibitors of influenza virus hemagglutinin (HA)-mediated membrane fusion that has a narrow and defined structure-activity relationship was identified. In Madin-Darby canine kidney (MDCK) cells infected with different strains of human influenza virus A/H3N2, the lead compound, 4c, displayed a 50% effective concentration of 3 to 23 muM and an antiviral selectivity index of 10. No activity was observed for A/H1N1, A/H5N1, A/H7N2, and B viruses. The activity of 4c was reduced considerably when added 30 min or later postinfection, indicating that 4c inhibits an early step in virus replication. 4c and its congeners inhibited influenza A/H3N2 virus-induced erythrocyte hemolysis at low pH. 4c-resistant virus mutants, selected in MDCK cells, contained either a single D112N change in the HA2 subunit of the viral HA or a combination of three substitutions, i.e., R220S (in HA1) and E57K (in HA2) and an A-T substitution at position 43 or 96 of HA2. The mutants showed efficiency for receptor binding and replication similar to that of wild-type virus yet displayed an increased pH of erythrocyte hemolysis. In polykaryon assays with cells expressing single-mutant HA proteins, the E57K, A96T, and D112N mutations resulted in 4c resistance, and the HA proteins containing R220S, A96T, and D112N mutations displayed an increased fusion pH. Molecular modeling identified a binding cavity for 4c involving arginine-54 and glutamic acid-57 in the HA2 subunit. Our studies with the new fusion inhibitor 4c confirm the importance of this HA region in the development of influenza virus fusion inhibitors.
鉴定出了一类新型 N-(1-硫代-4-氮杂螺[4.5]癸烷-4-基)甲酰胺类流感病毒血凝素（HA）介导的膜融合抑制剂，该抑制剂具有狭窄而明确的构效关系。在感染了不同株人季节性流感病毒 A/H3N2 的 Madin-Darby 犬肾（MDCK）细胞中，先导化合物 4c 的 50%有效浓度（EC50）为 3 至 23 μM，抗病毒选择性指数（SI）为 10。对 A/H1N1、A/H5N1、A/H7N2 和 B 型病毒均无活性。4c 在感染后 30 分钟或更晚添加时其活性明显降低，表明 4c 抑制病毒复制的早期步骤。4c 及其同类物可抑制流感 A/H3N2 病毒诱导的低 pH 下的红细胞溶血。在 MDCK 细胞中选择的 4c 抗性病毒突变体，要么在病毒 HA 的 HA2 亚基中仅有单个 D112N 改变，要么具有三个取代，即 HA1 中的 R220S 和 HA2 中的 E57K 以及 HA2 中位置 43 或 96 的 A-T 取代。这些突变体在受体结合和复制方面的效率与野生型病毒相似，但表现出红细胞溶血的 pH 值升高。在表达单突变 HA 蛋白的多核体测定中，E57K、A96T 和 D112N 突变导致 4c 耐药，并且包含 R220S、A96T 和 D112N 突变的 HA 蛋白显示出融合 pH 值升高。分子建模确定了 4c 的结合腔，涉及 HA2 亚基中的精氨酸-54 和谷氨酸-57。我们对新型融合抑制剂 4c 的研究证实了该 HA 区域在开发流感病毒融合抑制剂中的重要性。
