Influenza A viruses (IAVs) have gradually developed resistance to FDA-approved drugs, which increases the need to discover novel antivirals with new mechanisms of action. Here, we used a phenotypic screening strategy and discovered that the imidazo[1,2-a]pyrazine derivative A4 demonstrates potent and broad-spectrum anti-influenza activity, especially for the oseltamivir-resistant H1N1/pdm09 strain. Indirect immunofluorescence assays revealed that A4 induces clustering of the viral nucleoprotein (NP) and prevents its nuclear accumulation. Furthermore, upon conducting binding analyses between A4 and the influenza NP using surface plasmon resonance assays and molecular docking simulations, we were able to confirm that A4 binds directly to the viral NP. Additionally, A4 exhibits high human plasma metabolic stability (remaining_120 min > 90%, T_1/2 = 990 min) and moderate inhibitory effects on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 as well as low acute toxicity in Kunming mice. Overall, this study provides valuable insights and lays the groundwork for future efforts in medicinal chemistry to identify effective drugs against influenza.

中文翻译：

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鉴定咪唑并[1,2-a]吡嗪衍生物 A4 作为潜在的流感病毒核蛋白抑制剂

甲型流感病毒 (IAV) 已逐渐对 FDA 批准的药物产生耐药性，这增加了发现具有新作用机制的新型抗病毒药物的需求。在这里，我们使用表型筛选策略，发现咪唑并[1,2- a ]吡嗪衍生物A4表现出有效和广谱的抗流感活性，特别是对于奥司他韦耐药的H1N1/pdm09菌株。间接免疫荧光测定表明，A4诱导病毒核蛋白 (NP) 聚集并防止其核积聚。此外，在使用表面等离子共振测定和分子对接模拟对A4和流感 NP 之间进行结合分析后，我们能够确认A4直接与病毒 NP 结合。此外，A4在昆明小鼠中表现出较高的人血浆代谢稳定性（剩余_{120 分钟}> 90%，T _1/2 = 990 分钟），对 CYP1A2、CYP2C9、CYP2C19、CYP2D6 和 CYP3A4 具有中等抑制作用，且急性毒性较低。总的来说，这项研究提供了宝贵的见解，并为未来在药物化学领域寻找有效的流感药物奠定了基础。