## 4 Discussion  
Therapeutics are needed to help protect the population and to better prepare for future coronavirus outbreaks. In this study, Z-Tyr-Ala-CHN\({}_{2}\) was identified as a singleton in a primary phenotypic screen with antiviral activity against SARS-CoV-2 in VeroE6-eGFP cells. Available analogues of Z-Tyr-Ala-CHN\({}_{2}\) did not have any antiviral activity in VeroE6-eGFP, potentially because they all lack the diazo carbonyl group (RC(O)CH = N\({}^{+}\) = N\({}^{-}\)) motif that is present in Z-Tyr-Ala-CHN\({}_{2}\), a moiety that might act as a covalent binder to its target. Further research is warranted to assess if this moiety is indeed responsible for its activity, and whether the presumed covalent binding is due to the carbonyl function or the diazo group (or both). If this proves to be the case, it might be worth performing a diverse chemical space exploration maintaining the diazo carbonyl group, or to explore other covalent binder motifs, to improve compound potency. Indeed, covalent binders have proven valuable within various disease areas, with such binding motifs being present in many marketed drugs, a recent example being the SARS-CoV-2 M\({}^{\text{PRO}}\) inhibitor nirmatrelvir [53]. In viral yield reduction assays, Z-Tyr-Ala-CHN\({}_{2}\) had sub-micromolar activity (EC\({}_{50}\) = 0.37 uM) and caused a 4-log drop in virus replication without any signs of toxicity. The observed activity of Z-Tyr-Ala-CHN\({}_{2}\) was comparable to remdesivir and PF-00835231, two compounds with well-described antiviral activity against SARS-CoV-2 [44; 54]. As the COVID-19 pandemic progressed, new variants were detected on a regular basis [55]. Moreover, previous outbreaks with other coronavirus such as SARS-CoV-1 and Middle East respiratory syndrome coronavirus resulted in more serious illnesses [20]. Therefore, antivirals with broad-spectrum coronavirus activity are desirable. Z-Tyr-Ala-CHN\({}_{2}\) has activity against SARS-CoV-2, SARS-CoV-1, and HCoV-229E. Moreover, in both HeLa-hACE2 and A549-hACE2 cells, Z-Tyr-Ala-CHN\({}_{2}\) was more potent than remdesivir, PF-00835231, and molnupiravir against all coronaviruses tested, but was roughly ten times less potent than K777. Through TOA assays, it was established that Z-Tyr-Ala-CHN\({}_{2}\) acts in the early steps of the SARS-CoV-2 replication cycle, similarly to the cathepsin L inhibitor K777 and the PIKfive kinase blocker apilimod [29; 56; 57]. A pseudotyped virus neutralization assay in VeroE6 cells confirmed an entry mode of action for Z-Tyr-Ala-CHN\({}_{2}\). Camostat mesylate, a TMPRSS2 blocker, was not active in the pseudotyped virus neutralization assay in VeroE6 cells but was active when the assay was repeated using TMPRSS2-expressing VeroE6 cells. This demonstrates that in VeroE6 cells, entry is not dependent on TMPRSS2, whereas in the TMPRSS2-expressing cells, the TMPRSS2 pathway is preferred. Interestingly, Z-Tyr-Ala-CHN\({}_{2}\) was not active in TMPRSS2-expressing VeroE6 cells, suggesting that Z-Tyr-Ala-CHN\({}_{2}\) blocks entry via the endosomal pathway rather than via the TMPRSS2 pathway. Similarly, the cathepsin L inhibitor K777 lost most of its activity when tested on TMPRSS2-expressing VeroE6 cells. This observation is in line with previous studies on SARS-CoV-1 and SARS-CoV-2 [29; 58; 59; 60]. One of these studies shows lower activity of K777 in TMPRSS2-expressing Calu-3 and Caco-2 cells than in VeroE6 or HeLa/ACE2 cells [29]. Another study observes lower activity of K777 upon TMPRSS2 expression in A549-hACE2 cells [58]. Yet another study in which 293T-ACE2 cells were infected with SARS-CoV-2 pseudotyped virus demonstrates that the inhibitory activity of the cathepsin L blocker E-64d nearly vanishes upon TMPRSS2 expression [22]. Based on our neutralization assay results, as well as on these previous studies, it becomes clear that overexpression of TMPRSS2 in non-TMPRSS2-expressing cells abolishes dependence on the cathepsin L pathway for SARS-CoV-2 entry and restores sensitivity to TMPRSS2 blockers.