In Silico Analysis and Synthesis of Nafamostat Derivatives and Evaluation of Their Anti-SARS-CoV-2 Activity

Abstract

Inhibition of transmembrane serine protease 2 (TMPRSS2) is expected to block the spike protein-mediated fusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nafamostat, a potent TMPRSS2 inhibitor as well as a candidate for anti-SARS-CoV-2 drug, possesses the same acyl substructure as camostat, but is known to have a greater antiviral effect. A unique aspect of the molecular binding of nafamostat has been recently reported to be the formation of a covalent bond between its acyl substructure and Ser441 in TMPRSS2. In this study, we investigated crucial elements that cause the difference in anti-SARS-CoV-2 activity of nafamostat and camostat. In silico analysis showed that Asp435 significantly contributes to the binding of nafamostat and camostat to TMPRSS2, while Glu299 interacts strongly only with nafamostat. The estimated binding affinity for each compound with TMPRSS2 was actually consistent with the higher activity of nafamostat; however, the evaluation of the newly synthesized nafamostat derivatives revealed that the predicted binding affinity did not correlate with their anti-SARS-CoV-2 activity measured by the cytopathic effect (CPE) inhibition assay. It was further shown that the substitution of the ester bond with amide bond in nafamostat resulted in significantly weakened anti-SARS-CoV-2 activity. These results strongly indicate that the ease of covalent bond formation with Ser441 in TMPRSS2 possibly plays a major role in the anti-SARS-CoV-2 effect of nafamostat and its derivatives.

A Quantum Chemical Study on the Anti-SARS-CoV-2 Activity of TMPRSS2 Inhibitors

“…The ratios were calculated to be 7.59×10 4 , 3.04×10, and 1.65×10 −12 for nafamostat, camostat, and compound 1, respectively, indicating that the acyl intermediate is more likely to be formed with nafamostat and camostat, and less likely to be formed with compound 1. These thermodynamic analyses successfully reproduced a trend in the inhibitory activities of the three compounds which was obtained from in vitro studies 20 .…”

“…The calculations indicated the nafamostat derivative where the ester bond of nafamostat was replaced with an amide bond (compound 1) as having the highest binding affinity. Based on this result, we expected compound 1 exhibiting the strongest inhibitory activity 20 ; however, in vitro assays using SARS-CoV-2-infected cells demonstrated that compound 1 had the weakest inhibitory activity 20 .…”

“…We now discuss the relationship between the results of the previous in vitro assay 20 and this energy profile. As stated previously, nafamostat possesses stronger anti-SARS-CoV-2 activity than camostat 14 .…”

“…To investigate the differences in the potency of TMPRSS2 inhibitors, we previously calculated the binding affinity of nafamostat, camostat, and five nafamostat derivatives for TMPRSS2 20 . Notably, our calculations characterized the binding between TMPRSS2 and the compounds as a non-covalent interaction because drug potency is generally discussed in terms of binding affinity to the target protein via the classical treatment.…”

“…In other words, the ease of covalent bond formation with TMPRSS2 may contribute to the inhibitory activity. The aforementioned binding affinity factors in the interaction energy between the compound and TMPRSS2 when forming a non-covalent complex 20 . In contrast, the formation of covalent bonds corresponds to the occurrence of a chemical reaction and strongly depends on the relative energy of the product to the reactant and the activation energy in the chemical reaction 24 .…”