In Silico and In Vitro Evaluation of Some Amidine Derivatives as Hit Compounds towards Development of Inhibitors against Coronavirus Diseases

Abstract

Coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV and influenza A virus, require the host proteases to mediate viral entry into cells. Rather than targeting the continuously mutating viral proteins, targeting the conserved host-based entry mechanism could offer advantages. Nafamostat and camostat were discovered as covalent inhibitors of TMPRSS2 protease involved in viral entry. To circumvent their limitations, a reversible inhibitor might be required. Considering nafamostat structure and using pentamidine as a starting point, a small set of structurally diverse rigid analogues were designed and evaluated in silico to guide selection of compounds to be prepared for biological evaluation. Based on the results of in silico study, six compounds were prepared and evaluated in vitro. At the enzyme level, compounds 10-12 triggered potential TMPRSS2 inhibition with low micromolar IC₅₀ concentrations, but they were less effective in cellular assays. Meanwhile, compound 14 did not trigger potential TMPRSS2 inhibition at the enzyme level, but it showed potential cellular activity regarding inhibition of membrane fusion with a low micromolar IC₅₀ value of 10.87 µM, suggesting its action could be mediated by another molecular target. Furthermore, in vitro evaluation showed that compound 14 inhibited pseudovirus entry as well as thrombin and factor Xa. Together, this study presents compound 14 as a hit compound that might serve as a starting point for developing potential viral entry inhibitors with possible application against coronaviruses.

Keywords: antiviral agents; coronaviruses; viral entry.

Figure 1

Chemical structure of nafamostat (1), camostat (2), 3-amidinophenylalanyl-derived inhibitor (3), ketobenzothiazole-containing diguanidine inhibitor (4), pentamidine (5), propamidine (6), and eight investigated compounds as TMPRSS2 inhibitors (7–14).

Figure 2

Predicted binding modes and interactions of compounds (7–14) within the binding site of TMPRSS2 (PDB ID: 7MEQ): (A) predicted pose of compound 7 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2; (B) predicted pose of compound 8 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2; (C) predicted pose of compound 9 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2; (D) predicted pose of compound 10 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2; (E) predicted pose of compound 11 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2; (F) predicted pose of compound 12 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2; (G) predicted pose of compound 13 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2; (H) predicted pose of compound 14 (left) and schematic representation (right) of established interactions within the active site of TMPRSS2.

Scheme 1

Reagents and conditions: (a) (i) Na₂S₂O₅, EtOH-H₂O, reflux; (ii) NaOH/H₂O, EtOH/HCl.