## 3 Results
### Assessment of the Mechanism of Action of Z-Tyr-Ala-CHN\({}_{2}\)  
#### 3.5.1 Toa Assay  
The lack of Z-Tyr-Ala-CHN\({}_{2}\) activity in HNECs and Caco-2 cells demonstrates that there is a cell-specific antiviral effect and that it potentiates the involvement of a host target. To assess the mechanism of action of Z-Tyr-Ala-CHN\({}_{2}\), we determined the point at which Z-Tyr-Ala-CHN\({}_{2}\) affects the replication cycle of SARS-CoV-2 using an HCI-based TOA assay with intracellular dsRNA staining. The readout at 8 hpi was near the end of the first replication cycle but still before virion release [47; 48]. The TOA assay showed that Z-Tyr-Ala-CHN\({}_{2}\) antiviral activity was lost when administered at 2 h post virus infection or later, suggesting that the compound targets one of the early steps in the SARS-CoV-2 replication cycle (Figure 5A). A similar trend was seen for apilimod and K77, which were included as they block early steps in the virus replication cycle. In contrast, the inhibition of dsRNA formation by remdesivir, PF-07321332, and PF-00835231 was sustained until \(\sim\)4 hpi, confirming that these compounds exert an effect at a later point in time (a viral polymerase blocker and two M\({}^{\text{Pro}}\) inhibitors, respectively) [43; 44]. Figure 5B shows how the infection, measured using dsRNA staining, differs between Z-Tyr-Ala-CHN\({}_{2}\) and PF-07321332 across time points at which the compound was administered.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline  & & **Z-Tyr-Ala-CHN\({}_{2}\)** & **Remdesivir** & **PF-00835231** & **K777** & **Molnu-Piravir** \\ \hline EC\({}_{80}\)\(\pm\) SD (\(\upmu\)M) & Assay Type & & & & & \\ \hline VeroE6-eGFP & CPE reduction & & & & & \\ SARS-CoV-2 & (GFP) & 1.33 \(\pm\) 0.49 & 1.42 \(\pm\) 0.15 & \(>\)50 & 0.54 \(\pm\) 0.14 & NT \\ (\(n=2\)) & & & & & & \\ \hline A549-hACE2 & HCI & & & & & \\ SARS-CoV-2 & (spike stain) & 0.05 \(\pm\) 0.003 & 0.18 \(\pm\) 0.01 & 0.18 \(\pm\) 0.008 & 0.004 \(\pm\) 0.0002 & NT \\ (\(n=3\)) & & & & & & \\ \hline HeLa-hACE2 & HCI & & & & & \\ SARS-CoV-2 & (dsRNA stain) & 0.01 \(\pm\) 0.001 & 0.11 \(\pm\) 0.01 & 0.16 \(\pm\) 0.02 & 0.0006 \(\pm\) 0.00005 & 1.01 \(\pm\) 0.11 \\ (\(n=3\)) & & & & & & \\ \hline A549-hACE2 & HCI & & & & & \\ SARS-CoV-1 & (spike stain) & 0.05 \(\pm\) 0.016 & 0.33 \(\pm\) 0.02 & 0.55 \(\pm\) 0.04 & 0.003 \(\pm\) 0.0005 & NT \\ (\(n=3\)) & & & & & & \\ \hline HeLa-hACE2 & HCI & & & & & \\ HCO-2’29E & (dsRNA stain) & 0.07 \(\pm\) 0.01 & 0.11 \(\pm\) 0.009 & 0.47 \(\pm\) 0.04 & 0.002 \(\pm\) 0.0004 & 1.32 \(\pm\) 0.18 \\ (\(n=2\)) & & & & & & \\ \hline Caco-2 & CPE reduction & & & & & \\ SARS-CoV-2 & (manual scoring) & \(>\)50 & 0.77 \(\pm\) 0.26 & NT & NT & NT \\ (\(n=6\)) & & & & & & \\ \hline HNECs & vRNA & & & & & \\ SARS-CoV-2 & (qPCR) & \(>\)20 & NT & 0.18 \(\pm\) 0.14 \({}^{*}\) & NT & NT \\ (\(n=2\)) & & & & & & \\ \hline CC\({}_{50}\) (\(\upmu\)M) & Toxicity & & & & & \\ \hline A549-hACE2 & Toxicity & & & & & \\ (\(n=2\)-3) & (ATPlite) & & & & & \\ \hline VeroE6-eGFP & Toxicity & & & & & \\ (\(n=3\)) & (ATPlite) & & & & & \\ \hline SI & & & & & & \\ A549-hACE2 & & & & & & \\ SARS-CoV-2 & & & & & & \\ \hline \hline \end{tabular} \(n\) is the number of independent experiments on which the EC\({}_{50}\) or CC\({}_{50}\) calculations are based. \({}^{*}\) Oral Pfizer Mpro PF-07321332 was used instead of PF-00835231. CC\({}_{50}\), half-maximal cytotoxic concentration; CPE, cytopathic effect; dsRNA, double-stranded RNA; HCI, high-content imaging; EC\({}_{50}\), half-maximal effective concentration; NT, not tested; SD, standard deviation. SI, selectivity index; qPCR, quantitative polymerase chain reaction; vRNA, viral RNA.  
\end{table}
Table 1: EC\({}_{50}\) and CC\({}_{50}\) values \(\pm\) SD and SI values of antiviral agents tested by cellular background and virus type.