DEVELOPMENT OF DUAL INHIBITORS TARGETING THE VIRAL MAIN PROTEASE AND THE HOST CATHEPSIN L AS SARS-COV-2 ANTIVIRALS - PROJECT SUMMARY THE SEVERE ACUTE RESPIRATORY SYNDROME CORONAVIRUS 2 (SARS-COV-2), ALSO CALLED NOVEL CORONAVIRUS 2019 (NCOV-19), STARTED TO CIRCULATE AMONG HUMANS AROUND DECEMBER 2019, AND IT IS NOW WIDESPREAD AS A GLOBAL PANDEMIC. THERE IS NO VACCINE OR ANTIVIRAL AVAILABLE FOR SARS-COV-2. IN THIS GRANT, WE PROPOSE TO DEVELOP DUAL INHIBITORS TARGETING VIRAL MAIN PROTEASE AND CATHEPSIN L AS SARS-COV-2 ANTIVIRALS. USING THE FRET-BASED ENZYMATIC ASSAY, WE RECENTLY IDENTIFIED SEVERAL INHIBITORS INCLUDING BOCEPREVIR, GC-376, AND CALPAIN INHIBITORS II AND XII, THAT HAVE POTENT ACTIVITY WITH SINGLE-DIGIT TO SUBMICROMOLAR IC50 VALUES IN THE ENZYMATIC ASSAY. SIGNIFICANTLY, ALL FOUR COMPOUNDS INHIBIT INFECTIOUS SARS-COV-2 REPLICATION IN CELL CULTURE WITH EC50 VALUES RANGING FROM 0.5 TO 3.4 ΜM. OVERALL, THE COMPOUNDS IDENTIFIED PROVIDE PROMISING STARTING POINTS FOR THE FURTHER DEVELOPMENT OF SARS-COV-2 THERAPEUTICS. OUR DISCOVERY OF CALPAIN INHIBITOR II AS A POTENT INHIBITOR AGAINST SARS-COV-2 IS INNOVATIVE AS IT SUGGESTS IT MIGHT BE FEASIBLE TO DEVELOP SARS-COV-2 ANTIVIRALS BY SIMULTANEOUSLY TARGETING BOTH VIRAL MPRO AND HOST CATHEPSIN L, BOTH OF WHICH ARE ESSENTIAL FOR VIRAL REPLICATION. COMPARED TO RECENTLY REPORTED MPRO INHIBITORS, THE HITS IDENTIFIED FROM OUR STUDY REPRESENT THE MOST POTENT AND SELECTIVE DRUG CANDIDATES WITH A NOVEL MECHANISM OF ACTION, THEREFORE WARRANTING FURTHER DEVELOPMENT. GIVEN OUR ENCOURAGING PRELIMINARY DATA, WE PROPOSE TO OPTIMIZE DUAL INHIBITORS AS SARS-COV-2 ANTIVIRALS. THE OBJECTIVE OF THIS PROPOSAL IS TO DEVELOP DUAL INHIBITORS AS POTENT SARS-COV-2 ANTIVIRALS WITH HIGH POTENCY, SELECTIVITY, FAVORABLE PHARMACOKINETIC PROPERTIES, AS WELL AS BROAD-SPECTRUM ANTIVIRAL ACTIVITY AGAINST CLOSELY RELATED CORONAVIRUSES SUCH AS SARS AND MIDDLE EAST RESPIRATORY SYNDROME (MERS) CORONAVIRUSES. OUR GOALS OF THIS GRANT ARE TO IDENTIFY ADDITIONAL DUAL INHIBITORS THROUGH BOTH HIGH-THROUGHPUT SCREENING AND STRUCTURE-BASED LEAD OPTIMIZATION OF OUR RECENTLY IDENTIFIED DUAL INHIBITORS. BY TARGETING THE SARS-COV-2 MPRO, THE EXPECTED OUTCOMES OF THE PROPOSED RESEARCH ARE BROAD-ACTING CORONAVIRUS ANTIVIRALS WITH A CONFIRMED MECHANISM OF ACTION, A HIGH SELECTIVITY INDEX, AND FAVORABLE IN VITRO PHARMACOKINETIC PROPERTIES THAT ARE READY FOR IN VIVO ANTIVIRAL EFFICACY TESTING IN RELEVANT ANIMAL MODELS. OVERALL, THIS GRANT IS BASED ON STRONG PRELIMINARY DATA AND OUR EXPERTISE IN DEVELOPING ANTIVIRALS TARGETING CYSTEINE PROTEASES.