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The widespread coronavirus SARS-CoV-2 has already infected over 4 million people worldwide, with a death toll over 280,000. Current treatment of COVID-19 patients relies mainly on antiviral drugs lopinavir/ritonavir, arbidol, and remdesivir, the anti-malarial drugs hydroxychloroquine and chloroquine, and traditional Chinese medicine. There are over 2,118 on-going clinical trials underway, but to date none of these drugs have consistently proven effective. Cathepsin L (CatL) is an endosomal cysteine protease. It mediates the cleavage of the S1 subunit of the coronavirus surface spike glycoprotein. This cleavage is necessary for coronavirus entry into human host cells, virus and host cell endosome membrane fusion, and viral RNA release for next round of replication. Here we summarize data regarding seven CatL-selective inhibitors that block coronavirus entry into cultured host cells and provide a mechanism to block SARS-CoV-2 infection in humans. Given the rapid growth of the SARS-CoV-2-positive population worldwide, ready-to-use CatL inhibitors should be explored as a treatment option. We identify ten US FDA-approved drugs that have CatL inhibitory activity. We provide evidence that supports the combined use of serine protease and CatL inhibitors as a possibly safer and more effective therapy than other available therapeutics to block coronavirus host cell entry and intracellular replication, without compromising the immune system., Key messages • There are over 2118 on-going clinical trials for COVID-19 patients, but current results are limited. • Host cell membrane CatL and TMPRSS2 promote coronavirus cell entry by removing the virus surface spike protein S1 subunit. • In host cell endosomes, CatL removes the S1 subunit and promotes viral membrane fusion, viral RNA release, and replication. • At least 7 CatL-selective inhibitors effectively block coronavirus infection of human cells. • At least 10 US FDA-approved drugs exert CatL inhibitory activity. • Combined use of TMPRSS2 inhibitor camostat and CatL inhibitors or FDA-approved CatL inhibitory drugs merits consideration for urgent testing in COVID-19 patients. • Chloroquine-induced endosomal pH increase and protease inactivation inhibit coronavirus infection, but impair human adaptive immunity. • The protease inhibitor cocktail therapy proposed here could block coronavirus infection while sparing human adaptive immunity.

Human coronaviruses (HCoVs) enter cells via two distinct pathways: the endosomal pathway using cathepsins to activate spike protein and the cell-surface or early endosome pathway using extracellular proteases such as transmembrane protease serine 2 (TMPRSS2). We previously reported that clinical isolates of HCoV-229E preferred cell-surface TMPRSS2 to endosomal cathepsin for cell entry, and that they acquired the ability to use cathepsin L by repeated passage in cultured cells and were then able to enter cells via the endosomal pathway. Here, we show that clinical isolates of HCoV-OC43 and -HKU1 preferred the cell-surface TMRRSS2 to endosomal cathepsins for cell entry, similar to HCoV-229E. In addition, the cell-culture-adapted HCoV-OC43 lost the ability to infect and replicate in air-liquid interface cultures of human bronchial tracheal epithelial cells. These results suggest that circulating HCoVs in the field generally use cell-surface TMPRSS2 for cell entry, not endosomal cathepsins, in human airway epithelial cells., Highlights • Clinical isolates of HCoV-OC43 and -HKU1 were isolated from ALI-cultured HBTE cells. • Clinical isolates of HCoVs preferred the TMRRSS2 to cathepsins for cell entry. • Cell culture adapted HCoV-OC43 lost the ability to replicate in HBTE-ALI culture.