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)在该通路中具有关键作用,通过调节衔接蛋白2(AP2M1)μ亚基的磷酸化来调节CME。在这里,我们报告了一系列基于先前报道的1,2,4a的新型AAK1抑制剂,5-四氢-4H-苯并[b][1,4]恶嗪并[4,3-d][1,4]恶嗪支架。在23种合成化合物中,化合物12e是最有效的化合物,对AAK1的IC50值为9.38±0.34nM。使用涉及SARS-CoV-2假病毒感染hACE2-HEK293宿主细胞的模型评估12e对SARS-CoV-2的体外抗病毒活性。结果表明,与SGC-AAK1-1和LX9211相比,12e对SARS-CoV-2进入宿主细胞的体外抗病毒活性优越,其活性与相关和参考化合物8相当。机械上,所有AAK1抑制剂均减弱了AAK1诱导的AP2M1苏氨酸156的磷酸化,并破坏了AP2M1和ACE2之间的直接相互作用,最终抑制了SARS-CoV-2感染。值得注意的是,化合物8和12e在抑制AP2M1T156的磷酸化以及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12e在抑制SARS-CoV-2感染方面具有显着的功效,并有望成为开发针对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者。
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-4H-benzo[b] [1,4]oxazino[4,3-d] [1,4]oxazine scaffold. Among 23 synthesized compounds, compound 12e is the most potent one with an IC50 value of 9.38 ± 0.34 nM against AAK1. The in vitro antiviral activity of 12e against SARS-CoV-2 was evaluated using a model involving SARS-CoV-2 pseudovirus infecting hACE2-HEK293 host cells. The results revealed that 12e was superior in vitro 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 8. 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 8 and 12e exhibited a more potent effect in suppressing the phosphorylation of AP2M1 T156 and the interaction between AP2M1 and ACE2. In conclusion, novel AAK1 inhibitor 12e 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.