Coronavirus entry into host cells hinges on the interaction between the spike glycoprotein of the virus and the cell-surface receptor angiotensin-converting enzyme 2 (ACE2), initiating the subsequent clathrin-mediated endocytosis (CME) pathway. AP-2-associated protein kinase 1 (AAK1) holds a pivotal role in this pathway, regulating CME by modulating the phosphorylation of the μ subunit of adaptor protein 2 (AP2M1). Herein, we report a series of novel AAK1 inhibitors based on previously reported 1,2,4a,5-tetrahydro-4-benzo[] [1,4]oxazino[4,3-] [1,4]oxazine scaffold. Among 23 synthesized compounds, compound is the most potent one with an IC value of 9.38 ± 0.34 nM against AAK1. The antiviral activity of against SARS-CoV-2 was evaluated using a model involving SARS-CoV-2 pseudovirus infecting hACE2-HEK293 host cells. The results revealed that was superior antiviral activity against SARS-CoV-2 entry into host cells when compared to SGC-AAK1-1 and LX9211, and its activity was comparable to that of a related and reference compound . Mechanistically, all AAK1 inhibitors attenuated AAK1-induced phosphorylation of AP2M1 threonine 156 and disrupted the direct interaction between AP2M1 and ACE2, ultimately inhibiting SARS-CoV-2 infection. Notably, compounds and exhibited a more potent effect in suppressing the phosphorylation of AP2M1 T156 and the interaction between AP2M1 and ACE2. In conclusion, novel AAK1 inhibitor demonstrates significant efficacy in suppressing SARS-CoV-2 infection, and holds promise as a potential candidate for developing novel antiviral drugs against SARS-CoV-2 and other coronavirus infections.

中文翻译：

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新型 1,2,4a,5-四氢-4H-苯并[b][1,4]恶嗪基[4,3-d][1,4]恶嗪基 AAK1 抑制剂的设计、合成和生物学评价针对 SARS-CoV-2 的病毒特性


冠状病毒进入宿主细胞取决于病毒的刺突糖蛋白与细胞表面受体血管紧张素转换酶2（ACE2）之间的相互作用，启动随后的网格蛋白介导的内吞作用（CME）途径。 AP-2 相关蛋白激酶 1 (AAK1) 在此通路中发挥着关键作用，通过调节接头蛋白 2 (AP2M1) μ 亚基的磷酸化来调节 CME。在此，我们报道了一系列基于先前报道的1,2,4a,5-四氢-4-苯并[] [1,4]恶嗪基[4,3-] [1,4]恶嗪支架的新型AAK1抑制剂。在 23 种合成的化合物中，该化合物是最有效的一种，针对 AAK1 的 IC 值为 9.38 ± 0.34 nM。使用涉及 SARS-CoV-2 假病毒感染 hACE2-HEK293 宿主细胞的模型评估了针对 SARS-CoV-2 的抗病毒活性。结果表明，与 SGC-AAK1-1 和 LX9211 相比，该化合物对 SARS-CoV-2 进入宿主细胞具有优异的抗病毒活性，并且其活性与相关参考化合物相当。从机制上讲，所有 AAK1 抑制剂都能减弱 AAK1 诱导的 AP2M1 苏氨酸 156 磷酸化，并破坏 AP2M1 和 ACE2 之间的直接相互作用，最终抑制 SARS-CoV-2 感染。值得注意的是，化合物 和 在抑制 AP2M1 T156 磷酸化以及 AP2M1 和 ACE2 之间的相互作用方面表现出更有效的作用。总之，新型 AAK1 抑制剂在抑制 SARS-CoV-2 感染方面表现出显着功效，有望成为开发针对 SARS-CoV-2 和其他冠状病毒感染的新型抗病毒药物的潜在候选药物。