Evaluation of Novel Guanidino-Containing Isonipecotamide Inhibitors of Blood Coagulation Factors against SARS-CoV-2 Virus Infection

• Published in: Viruses 2022-08, Vol.14 (8), p.1730
• Main Authors: De Maio, Flavio, Rullo, Mariagrazia, de Candia, Modesto, Purgatorio, Rosa, Lopopolo, Gianfranco, Santarelli, Giulia, Palmieri, Valentina, Papi, Massimiliano, Elia, Gabriella, De Candia, Erica, Sanguinetti, Maurizio, Altomare, Cosimo Damiano
• Format: Article
• Language: English
• Subjects: ACE2; Angiotensin-converting enzyme 2; Animals; Anticoagulants; Anticoagulants (Medicine); Antithrombin; Blood coagulation; Blood Coagulation Factors; Chlorocebus aethiops; Coagulation factor Xa; Coronaviruses; COVID-19; COVID-19 Drug Treatment; Dabigatran; Dosage and administration; factor Xa; Guanidine; Health aspects; Humans; Infectivity; Laboratories; Mortality; Pharmacology, Experimental; Rivaroxaban; SARS-CoV-2; Serine; Serine proteinase; Severe acute respiratory syndrome coronavirus 2; Spike Glycoprotein, Coronavirus - metabolism; Spike protein; Statistical analysis; Testing; Thrombin; Vero Cells; Virus Internalization
• ISSN: 1999-4915; 1999-4915
• DOI: 10.3390/v14081730

Coagulation factor Xa (fXa) and thrombin (thr) are widely expressed in pulmonary tissues, where they may catalyze, together with the transmembrane serine protease 2 (TMPRSS2), the coronaviruses spike protein (SP) cleavage and activation, thus enhancing the SP binding to ACE2 and cell infection. In this study, we evaluate in vitro the ability of approved (i.e., dabigatran and rivaroxaban) and newly synthesized isonipecotamide-based reversible inhibitors of fXa/thr (cmpds - ) to hinder the SARS-CoV-2 infectivity of VERO cells. Nafamostat, which is a guanidine/amidine antithrombin and antiplasmin agent, disclosed as a covalent inhibitor of TMPRSS2, was also evaluated. While dabigatran and rivaroxaban at 100 μM concentration did not show any effect on SARS-CoV-2 infection, the virus preincubation with new guanidino-containing fXa-selective inhibitors and did decrease viral infectivity of VERO cells at subtoxic doses. When the cells were pre-incubated with , a reversible nanomolar inhibitor of fXa ( = 15 nM) showing the best in silico docking score toward TMPRSS2 (pdb 7MEQ), the SARS-CoV-2 infectivity was completely inhibited at 100 μM ( < 0.0001), where the cytopathic effect was just about 10%. The inhibitory effects of on SARS-CoV-2 infection was evident (ca. 30%) at lower concentrations (3-50 μM). The covalent TMPRSS2 and the selective inhibitor nafamostat mesylate, although showing some effect (15-20% inhibition), did not achieve statistically significant activity against SARS-CoV-2 infection in the whole range of test concentrations (3-100 μM). These findings suggest that direct inhibitors of the main serine proteases of the blood coagulation cascade may have potential in SARS-CoV-2 drug discovery. Furthermore, they prove that basic amidino-containing fXa inhibitors with a higher docking score towards TMPRSS2 may be considered hits for optimizing novel small molecules protecting guest cells from SARS-CoV-2 infection.