## 3 Results
### R428 and EMD Represent Promising Antiviral Candidates against Lassa Fever  
Our results, so far, revealed a cooperative inhibitory effect of EMD and R428 against LASV entry in the presence or absence of functional DG, by affecting macropinosome trafficking. Antiviral cocktails to combat RNA virus infection are especially powerful, when cocktails contain combinations of drugs targeting different steps of the viral cycle [101; 102; 103]. To date, the off-label administration of Rib is the only available treatment against LASV infections [4]. To investigate the antiviral activity of RTKs in combination with Rib, we pretreated A549 and HT-1080 cells with indicated drugs for 30 min at 37 \({}^{\circ}\)C. Upon drug preincubation, to synchronize infections, cells were quickly cooled down to 4 \({}^{\circ}\)C and infected at 4 \({}^{\circ}\)C with rLCMV/LASV\({}_{\text{GP}}\) in the presence of increasing concentrations of Rib (0-20 \(\upmu\)M), either alone (DMSO) or in combination with EMD (5 \(\upmu\)M) and increasing concentrations of R428 (0-1 \(\upmu\)M), and shifted to 37 \({}^{\circ}\)C, for 1 h. After infection, cells were incubated for 16-20 h at 37 \({}^{\circ}\)C in the presence of indicated drug concentrations and 20 mM of ammonium chloride. The results showed that R428 and EMD increased the antiviral activity of low to mild doses of Rib in both cell lines in the absence of overall toxicity (Figure 7 and Figure S6A-C).  
In agreement with previous results, the effect was more pronounced in HT-1080 than in A549 cells. To further determine the type of interaction, antagonistic, additive or synergistic, among the drugs present in the cocktail, we performed further analysis with CompuSyn [95]. In A549 cells, all R428/EMD and Rib combinations rendered CI values \(<1\), suggesting synergistic interaction between the RTK inhibitors and Rib (Table 3).  
\begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**A549 Cells**} \\ \hline
**Rib Concentration (\(\upmu\)M)** & **R428 + EMD (5 \(\upmu\)M) Concentration (\(\upmu\)M)** & **Effect** & **CI Value** \\ \hline
2.5 & 0.125 & 0.47 & 0.14113 \\ \hline
2.5 & 0.25 & 0.51 & 0.23307 \\ \hline
2.5 & 0.5 & 0.6 & 0.31581 \\ \hline
2.5 & 1.0 & 0.64 & 0.52867 \\ \hline
5.0 & 0.125 & 0.56 & 0.09568 \\ \hline \hline \end{tabular}
\end{table}
Table 3: Drug interaction (R428 + EMD/Rib) analysis in A549 cells. Analysis performed with CompuSyn [95]. CI values \(<1\) means synergistic interaction, approximately 1 additive effect, and \(>\)1 antagonistic interaction.  
Figure 7: Antiviral effect of R428, EMD, and Rib cocktails. A549 and HT-1080 cells were pretreated for 30 min, at 37 \({}^{\circ}\)C, cooled down to 4 \({}^{\circ}\)C, and infected with rLCMV/LASV\({}_{\text{GP}}\) in the presence of indicated concentrations of Rib, either in combination with DMSO or EMD (10 \(\upmu\)M) and R428 (0–1 \(\upmu\)M). Then, cells were moved to 37 \({}^{\circ}\)C, for 1 h. Upon infection, cells were incubated for 16–20 h in the presence of indicated drugs and 20 mM of ammonium chloride. Relative entry values were normalized to the infectivity in absence of any drug. Error bars represent standard deviations (\(n=3\)).  
In HT-1080 cells, moderate R428/EMD cocktail concentrations (R428 0.125-1 uM + EMD 5 uM) interacted synergistically with Rib, whereas low and high cocktail concentrations were closer to the additive effect (CI values approximately 1, Table 4).