# AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway
## 2 Results
### AG1478 Had a Superior Safety Profile as Compared to GCA and BFA  
As two existing GBF1 inhibitors (BFA and GCA) demonstrated different inhibitory characteristics as compared to AG1478 (Figure 4E), we decided to compare these three compounds for their antiviral activities. All compounds demonstrated decent antiviral activity (Figure 5A-C). However, AG1478 induced very little cellular toxicity across the entire dose range (1, 5, 10 uM) as demonstrated by a cell viability assay. In contrast, significant toxicity was observed for BFA and GCA even at 1 uM (Figure 5D). For GCA, its viral inhibition appeared to be mostly caused by cell death (Figure 5B).  
Figure 4: AG1478 targets GBF1. (**A**) Cells were transfected with either GBF1-GFP (overexpression) or GFP plasmid alone (control). GBF1 protein expression was detected by Western blot analysis. ACTIN was used as a loading control. (**B**) Viral titers were quantified by plaque assay in GBF1 overexpressed or GFP expressed cells. Triplicates were used for each experiment and experiments were repeated at least three times. Data shown are mean \(\pm\) SEM. \(n\) = 4, * and #: \(p\) < 0.05. (**C**) GBF1 siRNA (siGBF1) was used to knock down GBF1. Cells transfected with siRNA with a random sequence (SiC) were used as control. (**D**) Viral titers were quantified by plaque assay in siGBF1 or siC-transfected cells. (**E**) Cells were treated with AG1478, BFA, and GCA, and ARF1-GTP was pulled down. Total ARF1 protein was used as a loading control. This is a representative image from three independent repeats. (**F**) Cells were treated with AG1478 for 24 h, and then they were fixed and stained for GBF1 (Red). AG1478-containing media were removed from some of the wells and replaced with new media without AG1478 at 1 h before fixation (labeled as wash). DAPI (blue) was used for the nuclear staining. Images were taken under 63\(\times\) magnification. Scale bar: 5 uM.  
Figure 5: AG1478 targeted on GBF1. Beas2B cells were treated with (**A**) BFA at 1 and 10 \(\upmu\)M, (**B**) GCA at 1 and 10 \(\upmu\)M, and (**C**) AG1478 at 2 and 10 \(\upmu\)M and infected with A/WSN/33 influenza virus. (**D**) MTT assay was used to test cell viability after 24 h treatments of BFA, GCA, and AG1478 at indicated doses. Quadruplicates were used for each experiment and experiments were repeated at least three times. Data shown are mean \(\pm\) SEM. \(n=4\), #: \(p<0.05\). (**E**) The 1 and 24 h treatment of BFA, GCA, and AG1478. GM130 (green, a Golgi marker) and GBF1 (red) were measured by immunofluorescence. DAPI (blue) was used for the nuclear staining. Co-staining was shown in a combined image. Images were taken under 63\(\times\) magnification. Scale bar: 5 \(\upmu\)m. All were representative images from three independent repeats.  
As GBF1 activity is essential to maintain Golgi integrity, we tested how these three chemicals affected Golgi by fluorescence microscopy. In contrast to BFA and GCA that induced persistent Golgi dispersion, AG1478 only caused transient Golgi dispersion and it was fully recovered within 24 h (Figure 5E). Combining Figures 4E and 5, the antiviral activities of these three GBF1 inhibitors appeared not to be proportional to their effects on Golgi dispersion as well as to their cellular toxicity. Nevertheless, AG1478 demonstrated a low cellular toxicity, a mild effect on Golgi structure, and a potent antiviral activity. Thus, AG1478 is clearly the top candidate for antiviral drug development among these three chemicals.