key: cord-0312471-9rprzbi3 authors: Augusto Chaves, Otávio; Sacramento, Carolina Q.; Fintelman-Rodrigues, Natalia; Temerozo, Jairo Ramos; Pereira-Dutra, Filipe; Mizurini, Daniella M.; Monteiro, Robson Q.; Vazquez, Leonardo; Bozza, Patricia T.; Castro-Faria-Neto, Hugo Caire; Souza, Thiago Moreno L. title: Apixaban, an orally available anticoagulant, inhibits SARS-CoV-2 replication by targeting its major protease in a non-competitive way date: 2021-09-24 journal: bioRxiv DOI: 10.1101/2021.09.23.461605 sha: af10d40da2fbf6942cc438ed3899e125ac632965 doc_id: 312471 cord_uid: 9rprzbi3 Anticoagulants are associated with clinical benefit against the 2019 coronavirus disease (COVID-19), preventing COVID-19 associated coagulopathy. Blood coagulation factor Xa (FXa) and SARS-CoV-2 major protease (Mpro) share over 80% homology at the three-dimensional protein level. Thus, it is worth interrogating whether there is crosstalk between inhibitors and substrates between these enzymes. Here, we found that the clinically-approved FXa inhibitor apixaban targets SARS-CoV-2 Mpro with a 21-fold higher potency than boceprevir (GC376). Apixaban displayed a non-competitive mechanism of inhibition towards Mpro, since it targets the enzyme/substrate complex and the allosteric site onto the viral protease. Enzymatic assays were further validated in infected Calu-3 cells, which reveal that apixaban decreases the production of infectious viral particles in a dose-dependent manner, with an inhibitory potency in the micromolar range. Our results are in line with the proposed early use of anticoagulants, including FXa inhibitors, to improve clinical outcome of COVID-19 patients. In this context, apixaban may display a dual mechanism of action by targeting FXa to prevent coagulopathy and, at some level, SARS-CoV-2 Mpro. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the The main protease (M pro ) of SARS-CoV-2 is considered as one of the main 82 targets for drug repurposing, due to its cleavage activity at eleven sites at the viral Of note, the FXa inhibitor, ribaroxaban and the thrombin inhibitor, dabigatran were as 108 potent as boceprevir (Fig. 1C ). Since apixaban inhibits M pro with a Ki lower than the concentration of the viral 110 protease used in the assay, a non-canonical mechanism of inhibition over this enzyme 111 might be expected. When apixaban's inhibition over M pro was assayed under different 112 concentrations of substrate, a non-competitive mechanism was observed (Fig. 1D) . The Michaelis-Menten constant (Km) value was not altered by apixaban, suggesting M pro is 114 not halted to interact with its substrate by apixaban (Fig. 1D ). In addition, there was a 115 significant decrease in the M pro maximum velocity (Vmax) by apixaban (Fig. 1D) , 116 indicating that the enzyme (E) M pro bound to its substrate (S), in the enzyme-substrate 117 complex (ES) and was unable to cleave it and form the products (P) (Fig. 1E ). Based on molecular docking, we explored different alternative to explain these 119 results. When interacting with M pro , its peptidic substrate occupies the four enzymatic 120 subsites (S1, S1', S2, and S4) onto the active binding pocket with a docking score 121 (dimensionless) of 65.54 (Fig. 1F ). In the M pro /peptide (ES) complex, the substrate 122 forms an external loop, which is targeted by the anticoagulants (Fig. 1G ); in particular, 123 apixaban has the most favorable interaction compared with dabigatran and rivaroxaban approximately 3-fold more potent than any other anti-clotting drugs tested (Table 1) . 149 Nevertheless, apixaban was about 5-and 60-fold less potent in vitro in comparison to 150 the positive control atazanavir and remdesivir, respectively ( Table 1) , indicating that 151 apixaban shows an interestingly scaffold for the design of novel compounds to increase 152 6 its antiviral action. Despite a slightly higher cytotoxicity, compared to other tested 153 compounds, apixaban's selectivity index (SI) for SARS-CoV-2 replication was two-fold 154 better than other anticoagulants (Table 1) . Altogether our results confirm that 155 apixaban's chemical structure is endowed with antiviral activity against SARS-CoV-2. bound to albumin 30 , its free fraction at Cmax is equivalent to 72 nM, almost 10-times 187 higher than apixaban's Ki towards M pro . On the other side, cell-based assays, display 188 apixaban's potency as 3-times higher than human Cmax. Despite apixaban is more potent 189 than atazanavir to inhibit M pro , the IC50 value for apixaban was not better than A new coronavirus associated with human respiratory disease in China Subcommittee on disseminated intravascular coagulation. 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