Figure 5.

UPCDC-30245 inhibits HCoV-229E infection via blocking virus entry in H1299 cells. (A) Schema of time-of-addition experiment. 1. Added compound for 5 min, added virus for 2 h, washed out, added fresh media with DMSO for 6 h. 2. Added compound and virus together for 2 h, washed out, added fresh media with compound for 2 h, washout, add fresh media and DMSO for 4 h. 3. Added compound and virus together for 2 h, washed out, added fresh media with compound for 6 h. 4. Added DMSO and virus together for 2 h, washed out, added fresh media with DMSO for 2 h, washed out, and added fresh media with compound for 4 h. (B) The HCoV-229E RNA levels in H1299 cells were significantly reduced by 5 μM of UPCDC-30245 and 50 μM of HCQ at 8 h post infection. Data were normalized to the RNA levels of human GAPDH. Numbers 1 to 4 represent the time-of-addition described in Figure 5A. N = 3. (C) The protection of CPE activity (black curve) and toxicity (TOX, red curve) curves for UPCDC-30245 and HCQ. N = 4. (D) TCID₅₀/mL measurements in a viral titer reduction assay for DMSO, UPCDC-30245 and 25 μM of HCQ after 24 h of HCoV-229E infection. N = 3. (E) TCID₅₀/mL measurements for remdesivir. DMSO or 2.5 μM of UPCDC-3245 was added 5 min before virus infection and washed out with virus after 4 h of infection, then remdesivir was added and incubated for 20 h. N = 3. (F) The protection of CPE activity curves for remdesivir (0–0.42 μM, 3-fold dilution) with or without the pretreatment of 2.5 μM of UPCDC-30245 during virus infection. N = 3. ns, not significant; *, p < 0.05; **, p < 0.01; ****, p < 0.0001 according to one-way ANOVA with multiple comparison tests.