## 3 Results
### Assessment of the Mechanism of Action of Z-Tyr-Ala-CHN\({}_{2}\)
#### 3.5.3 Z-Tyr-Ala-CHN\({}_{2}\)  
Is a Cathepsin L Inhibitor That Does Not Cause Disruption of Lysosome Trafficking  
Since Z-Tyr-Ala-CHN\({}_{2}\) acts early in the SARS-CoV-2 replication cycle and activity was lost in the pseudotyped virus neutralization assay when TMPRSS2 was present, it is probable that the compound acts within the endolysosomal entry pathway. Moreover, the well-described cathepsin L inhibitor K777, had a similar activity profile in both the TOA assay and pseudovirus assay. Since cathepsin L is an important enzyme in the endolysosomal entry pathway, and because previous studies, though limited in number, have demonstrated that Z-Tyr-Ala-CHN\({}_{2}\) interacts with and inhibits cathepsin L and B [50; 51], we performed an enzymatic cathepsin L assay to assess whether Z-Tyr-Ala-CHN\({}_{2}\) acts on cathepsin L. Like the positive control leupeptin and the known cathepsin L blocker K777, Z-Tyr-Ala-CHN\({}_{2}\) blocked cathepsin L activity with an EC\({}_{50}\) < 0.05 uM (Figure S3).  
It is known that some compounds exert antiviral activity by impairing the endolysosomal pathway in an untargeted manner [52]. As this is undesirable, we wanted to assess whether Z-Tyr-Ala-CHN\({}_{2}\) impairs lysosomal trafficking. In this manner, we can determine whether its observed in vitro activity results from cathepsin L inhibition or instead results from untargeted disruption of the endolysosomal pathway. To this end, an HCI-based antiviral assay with LysoTracker dye was performed. PF-00835231, remdesivir, and K777 were included in the assay as reference compounds (Figure 7). Since remdesivir and the oral form of PF-00835231 are both approved antivirals for the treatment of COVID-19 infection, and act post entry, we did not expect them to disrupt lysosomal trafficking. Indeed, neither one of the compounds increased the intensity of the LysoTracker signal. K777 significantly increased the intensity of the LysoTracker signal at a concentration of \(\geq\)2.2 uM (adjusted \(p\)-value < 0.05). However, the significant increase only occurred at concentrations that are \(>\)1000 times higher than its EC\({}_{50}\) value of 0.0006 uM (\(n\) = 4) retrieved from the same assay. Z-Tyr-Ala-CHN\({}_{2}\) did not significantly increase the intensity of the LysoTracker signal, not even at the highest test concentrations. Hence, the antiviral effect against SARS-CoV-2 of Z-Tyr-Ala-CHN\({}_{2}\) was not deemed to be resulting from a disruption in lysosome trafficking and instead can be attributed to cathepsin L inhibition.  
Figure 7: HCI assay with LysoTracker to compare organelle accumulation between different compounds in HeLa-hACE2 cells. The dotted horizontal lines represent the mean \(\pm\)3 \(\times\) SD of the LysoTracker signal from the virus control. * is indicative for significant organelle accumulation (adjusted \(p\) < 0.05) at the corresponding concentration relative to the virus control, as determined by using two-way analysis of variance with Sidák correction for multiple comparisons. The intra-experiment dependency of the data is taken into account. The mean values \(\pm\) SD are based on four experiments.