Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science The University of Sydney Sydney NSW2006 Australia

Center for Marine Biotechnology and Biomedicine Scripps Institution of Oceanography University of California San Diego La Jolla California92093 United States

Charles Perkins Centre and School of Life and Environmental Sciences The University of Sydney Sydney NSW2006 Australia

Department of Biochemistry and Biophysics Texas A and M University 301 Old Main Drive College Station Texas77843 United States

Department of Microbiology and Immunology University of Texas Medical Branch 3000 University Boulevard Galveston Texas77755 1001 United States

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic 16610Prague Czech Republic

Kirby Institute University of New South Wales Sydney NSW2052 Australia

School of Chemistry The University of Sydney Sydney NSW2006 Australia

School of Medical Sciences Faculty of Medicine and Health The University of Sydney Sydney NSW2006 Australia

Skaggs School of Pharmacy and Pharmaceutical Sciences University of California San Diego 9500 Gilman Drive La Jolla California92093 United States

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Nature-Inspired Gallinamides Are Potent Antischistosomal Agents: Inhibition of the Cathepsin B1 Protease Target and Binding Mode Analysis