## 3 Results
### Assessment of the Mechanism of Action of Z-Tyr-Ala-CHN\({}_{2}\)
#### 3.5.2 Pseudotyped Virus Neutralization Assay in VeroE6 Cells  
In the TOA assay, we observed that Z-Tyr-Ala-CHN\({}_{2}\) is active early in the viral replication cycle. A pseudotyped virus neutralization assay was used to confirm an entry mode of action of Z-Tyr-Ala-CHN\({}_{2}\) and to assess whether Z-Tyr-Ala-CHN\({}_{2}\) acts on the endosomal entry route or on the TMPRSS2-dependent entry route. A Regeneron anti-spike monoclonal antibody was included as positive control as it can block the interaction between spike and hACE2, and thus can block entry independent of the entry route chosen by the pseudotyped virus. Like the Regeneron anti-spike monoclonal antibody, both Z-Tyr-Ala-CHN\({}_{2}\) and K77 could neutralize SARS-CoV-2 pseudovirus (SARS2 pp) entry in VeroE6 with EC\({}_{50}\) values of 0.13 and 0.023 uM, respectively (Figure 6, left panel). Camostat mesylate, a TMPRSS2 blocker, was included to assess which entry route was used by the pseudotyped virus to enter the cell. The compound was not active in the assay in VeroE6 (EC\({}_{50}>\) 30 uM) but was active (EC\({}_{50}\sim\)18 uM) when the assay was repeated in  
Figure 5: **(A)** TOA assay comparing the percentage inhibition of dsRNA formation in SARS-CoV-2-infected cells at 8 hpi by Z-Tyr-Ala-CHN\({}_{2}\), remdesivir, PF-07321332 (nirmatrelvir), PF-00835231, K777, and apilimod added at 0, 2, and 4 hpi. In one of the runs, 2.5 uM of compound was used, whereas the other two runs used 5 uM of compound; both concentrations are known to result in \(\sim\)100% inhibition of dsRNA in HeLa-hACE2 cells when added at 0 hpi. The mean values \(\pm\) SD are shown from two independent experiments for Z-Tyr-Ala-CHN\({}_{2}\), remdesivir, apilimod, and PF-00835231, and from three independent experiments for PF-07321332 and K777. The dotted horizontal lines represent the mean \(\pm\)3 \(\times\) SD of the virus control, indicative for the variation within the assay. **(B)** Microscopic images (20\(\times\) objective) of representative wells treated with Z-Tyr-Ala-CHN\({}_{2}\) or PF-07321332 at the indicated time points post infection. The Phaedra HCI analysis software was used to calculate cell outlines (white lines) using the CellMask Deep Red staining (channel not shown) and the nuclear staining (Hoechst 33342; blue). The dsRNA signal is shown in green. In the untreated/virus control condition, an average of 30% of the cells were dsRNA positive at 8 hpi. hpi, hours post infection; TOA, time of addition. VeroE6 TMPRSS2 cells in which SARS2 pp can enter via the TMPRSS2-dependent entry route (Figure 6, right panel). In contrast, the EC\({}_{50}\) of K77 shifted from 0.023 uM to 18.6 uM when repeated on VeroE6 TMPRSS2 cells, while Z-Tyr-Ala-CHN\({}_{2}\) was not active at all upon expression of TMPRSS2 in VeroE6. This observation suggests that Z-Tyr-Ala-CHN\({}_{2}\) acts on the endosomal entry pathway. None of the compounds induced toxicity at the concentrations tested, as can be seen from the CC\({}_{50}\) values in the figure. Figure 6: Pseudotyped SARS-CoV-2 neutralization assay in VeroE6 cells (left panel) and in VeroE6 TMPRSS2 cells (right panel). The Regeneron monoclonal antibody was included as a positive control because it can block entry in both cell types. VSV-Gpp was included to determine the specificity of the compounds. For infection with SARS2 pp and VSV-Gpp, the mean \(\pm\) SD values are shown for three to four independent experiments, whereas for cell viability, the mean \(\pm\) SD values are shown for two to three independent experiments. Mean EC\({}_{50}\) and CC\({}_{50}\) values of the different compounds in the pseudotyped SARS-CoV-2 neutralization assay are given as well. NT, not tested; TMPRSS2, transmembrane protease serine subtype 2; VSV-Gpp, vesicular stomatitis virus fusion glycoprotein G; SARS2 pp, SARS-CoV-2 S protein.