Fig. 1

The antiviral efficacy of PIKfyve inhibitors against SARS-CoV-2 and its variants of concern. a Structures of PIKfyve inhibitors and a reference cmpd 24. b The morphology of Vero E6 cells upon 5 μM drug treatment for 8 h. Scale bar, 50 µm. c Schematic of infectivity assay and the antiviral activity of indicated compounds against SARS-CoV-2. Vero E6 cells were infection with SARS-CoV-2 at MOI of 0.05. EC₅₀s were determined by qRT-PCR of virus gene (n = 3). CC₅₀s was determined by MTS assays (n = 3). Data are means ± SEM. The Graph is one representative result from three independent experiments. d The antiviral ability of PIKfyve inhibitors against four VOCs. Representative data from Fig. S3a. Data were shown as means ± SEM (n = 6). e Visualization of virus entry using STG probe. Cells were pretreated with 2 μM compounds for 1 h before STG addition. Confocal images of STG (green), ACE2-mRuby3 (red), and nucleus (blue) in 293T-ACE2iRb3 cells were taken at 3 h post STG incubation. Scale bar, 10 µm. f XMU-MP-7 inhibits cathepsin B activation. A549 cells were treated with DMSO or XMU-MP-7 for different time or concentrations as indicated. Cathepsin B and α-tubulin were analyzed by Western blot. g, h Time-of-addition experiment of PIKfyve inhibitors. The specific treatment of drugs and viruses at different stages was shown in the scheme. Viral replication was quantified by qRT-PCR at 24 h post infection (n = 3) (g). Nucleoprotein and GAPDH were analyzed by Western blot (h). NC, negative control, protein sample derived from cells that were not infected with virus. i PIKfyve inhibitors induce vacuolization in entry and post-entry stages. Vero E6 cells were treated with 2 μM compounds or DMSO for different time periods. Two hours after treatment, cells were washed twice with PBS and cultured in drug-free medium for another 48 h. Scale bar, 50 µm