## 4 Discussion
Some research suggests that compared with monotherapy, a combination therapy of a TMPRSS2 blocker and an endosomal pathway blocker could have several advantages, including synergistic antiviral efficacy, fewer side effects due to the lower amounts of drugs used, and potentially, a higher barrier to drug resistance [22; 23; 27; 60; 75]. For instance, camostat alone at 10 uM was shown not to fully block infection in TMPRSS2\({}^{+}\) Calu-3 cells, whereas the combination of 10 uM camostat with 10 uM EST, a broad inhibitor of cysteine proteases, could fully block the infection of SARS-CoV-1 [60]. Moreover, some recent studies suggest that the SARS-CoV-2 Omicron BA.1 and BA.2 variants, which together accounted for most of the new infections in the first half of 2022 [76], preferentially use the endosomal entry pathway instead of TMPRSS2-dependent cell surface entry [77; 78; 79]. This shift in entry was also described for Omicron infection in HNECs, cells that express both TMPRSS2 and cathepsin L [80]. Surprisingly, Z-Tyr-Ala-CHN\({}_{2}\) was unable to block entry of the SARS-CoV-2 Omicron BA.1 strain in the HNEC ALI cultures at concentrations up to 20 \(\upmu\)M, which suggests that, at least in this model, Omicron entry also makes use of the TMPRSS2 pathway. Even though in this work a small molecule screening approach was chosen that resulted in the identification of the cathepsin L inhibitor Z-Tyr-Ala-CHN\({}_{2}\), when the aim is to combat SARS-CoV-2 through cathepsin L inhibition, antibodies or peptide inhibitors might also hold promise as these can be highly potent and specific [81; 82]. Neutralizing antibodies targeting spike have, for instance, already proven to have potential as SARS-CoV-2 therapeutics with many in preclinical and clinical development, [83; 84] while several studies have thoroughly investigated peptide inhibitors as SARS-CoV-2 antivirals [85; 86]. Studies that further evaluate the in vivo potential of cathepsin L blockers are warranted. One in vivo study in African green monkeys demonstrated that the cathepsin L blocker K777 can limit SARS-CoV-2 pathogenesis and disease severity both in a prophylactic and therapeutic setting [26]. K777 has completed phase 1 clinical trials [29] and recruitment for a phase 2a trial is ongoing to assess its effect on clinical symptoms of COVID-19 infection (ClinicalTrials.gov identifier: NCT04843787). The outcome of this trial will teach us a great deal about the future role of cathepsin L blockers in the treatment of coronavirus infections. Aside from whether cathepsin L inhibitors have clinical potential against coronaviruses or not, Z-Tyr-Ala-CHN\({}_{2}\) can serve as a valuable tool compound to study coronavirus entry and replication as it has cell-specific activity, is highly potent, and does not show signs of toxicity. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/microorganisms11030717/s1](https://www.mdpi.com/article/10.3390/microorganisms11030717/s1)]([https://www.mdpi.com/article/10.3390/microorganisms11030717/s1](https://www.mdpi.com/article/10.3390/microorganisms11030717/s1)), Figure S1: Structure and activity data (EC\({}_{50}\) and CC\({}_{50}\)) of Z-Tyr-Ala-CHN\({}_{2}\) (n = 2) and its analogues (n = 1) derived from the primary screen in VeroE6-eGFP; Figure S2: Z-Tyr-Ala-CH\({}_{2}\) does not inhibit SARS-CoV-2 infection in an upper respiratory tract model. HNEC ALL cultures were infected with the indicated isolates 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%); Figure S3: Enzymatic cathepsin L assay demonstrating that both Z-Tyr-Ala-CHN\({}_{2}\) and K777 inhibit cathepsin L with EC\({}_{50}\) < 0.05 \(\upmu\)M. Leuepeptin, an inhibitor of endosomal trypsin-like serine and cysteine proteases, was included as a positive control, whereas water was included as a negative control. J.D.