## 3 Results
### Antiviral Activity of Z-Tyr-Ala-CHN\({}_{2}\) across Different Cellular Backgrounds  
To rule out cell-specific effects of Z-Tyr-Ala-CHN\({}_{2}\) against SARS-CoV-2, its antiviral activity was tested in A549-hACE2 and HeLa-hACE2 cells using intracellular staining of S protein or dsRNA, and in Caco-2 cells using visual CPE scoring. Z-Tyr-Ala-CHN\({}_{2}\) was found to potently inhibit infection in both A549-hACE2 and HeLa-hACE2 cells. However, no effect was observed in Caco-2 cells (Figure 4A; A549-hACE2: EC\({}_{50}\), 0.046 uM; CC\({}_{50}\), >25 uM; SI, >500; HeLa-hACE2: EC\({}_{50}\), 0.006 uM; Caco-2: EC\({}_{50}\), >50 uM; CC\({}_{50}\), >50 uM). To further investigate the cell dependency of the observed effect, we also assessed the activity of Z-Tyr-Ala-CHN\({}_{2}\) in a translational model of the upper respiratory tract. This model was established by growing HNECs in ALI [45] and by infecting the apical compartment of the culture with an early Belgian isolate (Lineage A, B.1) of SARS-CoV-2. Infection was monitored by measuring viral RNA copies in the apical culture supernatants for three days (Figure 4B). The M\({}^{\rm pro}\) inhibitor nitraturelvir/PF-07321332 was included as reference antiviral as intronicy-boosted nitraturelvir is currently the standard of care for mild to moderate COVID-19 infection [46]. Since it can be administered orally, it is a more relevant reference than remdesivir in this system. Z-Tyr-Ala-CHN\({}_{2}\) did not show antiviral activity at 20 uM whereas PF-07321332 at 2.5 uM did. Similarly, when the experiment was performed using the SARS-CoV-2 Omicron variant (B.1.1.529-BA.1), no inhibitory activity of Z-Tyr-Ala-CHN\({}_{2}\) was observed (Figure S2). Table 1 summarizes the activity of Z-Tyr-Ala-CH\({}_{2}\) across different coronaviruses and cellular backgrounds, as well as how it compared to remdesivir, K777, PF-00835231, and molinupiravir. K777 was included as it acts on an early step in the virus life cycle, namely, cathepsin L inhibition [29]. All the other drugs were included as they, or variants thereof, are on the market for treatment of COVID-19 infection. K777 showed the lowest EC\({}_{50}\) in HeLa-hACE2 and A549-hACE2 cells for all human coronaviruses tested (SARS-CoV-2, SARS-CoV-1, and HCoV-229E), followed by Z-Tyr-Ala-CHN\({}_{2}\) with over ten times lower potency than K777. Thus, in these cellular backgrounds, Z-Tyr-Ala-CHN\({}_{2}\) outperformed remdesivir, PF-00835231, and molinupiravir. Figure 3: Antiviral activity of Z-Tyr-Ala-CHN\({}_{2}\) against SARS-CoV-1 (black, full circles) in A549-hACE2 and against HCoV-229E (black, empty squares) in HeLa-hACE2. Infection of SARS-CoV-1 was detected using an anti-spike antibody, whereas infection against HCoV-229E was detected using an anti-dsRNA antibody. The mean \(\pm\) SD values are shown from two to three independent experiments. Cytotoxicity determined in uninfected A549-hACE2 using ATPlite (red, full triangles) represents the mean \(\pm\) SD of two independent experiments. HCoV-229E, human coronavirus 229E; SD, standard deviation; SI, selectivity index. Figure 4: **(A)** The antiviral activity of Z-Tyr-Ala-CHN\({}_{2}\) against SARS-CoV-2 in A549-hACE2 (black, empty squares), HeLa-hACE2 (black, full circles), and Caco-2 (black, full triangles) cells. The mean values \(\pm\) SD are shown of three (A549-hACE2 and HeLa-hACE2) to six (Caco-2) independent experiments. Cytotoxicity was determined in A549-hACE2 (red, empty squares) using ATPlite and in Caco-2 (red, full triangles) using Roitest® Vital. The mean values \(\pm\) SD are shown from two (A549-hACE2) to six (Caco-2) independent experiments. **(B)** Z-Tyr-Ala-CHN\({}_{2}\) does not inhibit SARS-CoV-2 infection in an upper respiratory tract model. HNEC ALI cultures were infected with SARS-CoV-2 and treated with PF-07321332 at 2.5 \(\upmu\)M (red) and Z-Tyr-Ala-CHN\({}_{2}\) at 20 \(\upmu\)M (black). Cell control (CC; (gray bars, DMSO 0.2%); Virus control (VC; blue bars, MOI 0.1 without compound).