Design, synthesis, and biological evaluation of novel AAK1/HDACs dual inhibitors against SARS-CoV-2 entry.

AP2-associated protein kinase 1 (AAK1) is a crucial regulator of clathrin-mediated endocytosis, involved in various cellular processes, including viral infection. Histone deacetylases (HDACs) are essential in regulating gene transcription through the process of histone deacetylation and have become promising therapeutic targets for the treatment of cancer and viral infections. In this study, several AAK1/HDACs dual inhibitors based on our previous reported compounds were designed and synthesized, and the antiviral activity of these dual inhibitors were evaluated. Among them, compound 12 showed remarkable dual inhibitory activity against both AAK1 and HDACs, with IC₅₀ values of 15.9 nM for AAK1, 148.7 nM for HDAC1, and 5.2 nM for HDAC6. Notably, this compound exhibited superior efficacy in suppressing SARS-CoV-2 entry into host cells compared to its close analogs 4, 13a, and 13b. Mechanistically, compound 12 attenuated AAK1-induced phosphorylation of adaptor protein-2 μ subunit (AP2M1) threonine 156, disrupting the direct interaction between AP2M1 and ACE2, thus inhibiting the CME-mediated SARS-CoV-2 endocytosis. Additionally, compound 12 increased the acetylation levels of H3K27 and α-tubulin, suggesting its potential as an epigenetic modulator. Overall, our findings propose compound 12 as a promising dual inhibitor against AAK1 and HDACs, highlighting its therapeutic potential in antiviral infections.