## 4 Discussion
Thus, in cell types where TMPRSS2 expression is high, cathepsin L utilization seems minimal, rendering poor efficacy of cathepsin L blockers. In line with the observed loss of activity in TMPRSS2-expressing VeroE6 cells, Z-Tyr-Ala-CHN\({}_{2}\) was also inactive against SARS-CoV-2 in Caco-2 cells, a cell type that endogenously expresses TMPRSS2 and that is sensitive to camostat mesylate [22]. Similarly, Z-Tyr-Ala-CHN\({}_{2}\) did not show antiviral activity in differentiated HNECs infected with SARS-CoV-2, whereas camostat mesylate did. This observation demonstrates that SARSCoV-2 entry in HNECs is TMPRSS2 dependent, and that endosomal fusion is limited or lacking. Due to the activity profiles of Z-Tyr-Ala-CHN\({}_{2}\) and the cathepsin L inhibitor K777 being similar in the TOA assay and in the pseudotyped virus neutralization assay, and because previous reports demonstrated that Z-Tyr-Ala-CHN\({}_{2}\) targets cathepsin L and B [50; 51], an enzymatic cathepsin L assay was performed to assess whether Z-Tyr-Ala-CHN\({}_{2}\) indeed targets cathepsin L, and thereby exerts its activity within the endosomal pathway. From this assay, it became clear that Z-Tyr-Ala-CHN\({}_{2}\) is a potent cathepsin L inhibitor with an EC\({}_{50}<0.05\) uM. Since Z-Tyr-Ala-CHN\({}_{2}\) acts on cathepsin L in the endosomal pathway, we assessed whether the compound could also disrupt the endolysosomal pathway in an untargeted manner, as this is a common, but less favorable mechanism of action of many coronavirus inhibitors [52; 61; 62; 63; 64; 65]. Since Z-Tyr-Ala-CHN\({}_{2}\) did not induce a significant increase in LysoTracker signal (reflective of low pH organelle accumulation), the compound's in vitro antiviral activity is most likely not a result of endolysosomal pathway disruption and instead can be attributed to cathepsin L inhibition. That said, it is worth considering if Z-Tyr-Ala-CHN\({}_{2}\) might exhibit dual activity with cathepsin L inhibition being most prominent. This is deemed unlikely since Z-Tyr-Ala-CHN\({}_{2}\) is relatively potent in cell types where SARS-CoV-2 depends on cathepsin L for entry but remains inactive at high compound concentration in cell types like HNECs and Caco-2 where TMPRSS2 is preferentially used for entry. Knowing that Z-Tyr-Ala-CHN\({}_{2}\) is a cathepsin L inhibitor also helps explain why the compound has broad-spectrum activity as, in addition to the S protein of SARS-CoV-1 and SARS-CoV-2, the HCoV-299E S protein can also be activated by TMPRSS2 and cathepsin L. Thus, in cell types where TMPRSS2 expression is minimal or lacking, like HeLa-hACE2, cathepsin L inhibition is expected to prevent infection of HCoV-229E. In line with our results, inhibition of HCoV-229E pseudovirus infection in 293T-CD13 cells was also possible using the cathepsin L blocker K777 [67; 68; 31]. It is unclear whether targeting cathepsin L can be a valuable therapeutic strategy against SARS-CoV-2 infection since TMPRSS2 expression can abolish dependence on the cathepsin L pathway for virus entry, and because TMPRSS2 is expressed in cell types in the airway that are targeted by SARS-CoV-2 [69; 70], namely ciliated cells in the upper respiratory tract and alveolar type 2 cells in the alveoli [71; 72]. It is, however, conceivable that the expression of cathepsin L relative to TMPRSS2 throughout the human airway epithelium also has an impact on the entry route. Interestingly, it is reported that cathepsin L protein and mRNA levels are increased in SARS-CoV-2-infected Huh-7 cells, while also, cathepsin L is significantly upregulated in both plasma levels and lungs of COVID-19 patients [73; 74]. Moreover, a limited number of in vivo studies exist that demonstrate that cathepsin L inhibition can prevent infection and limit SARS-CoV-2 pathogenesis [26; 73]. These findings suggest that cathepsin L could be an important factor during infection and disease progression.