Dual inhibitors of SARS-CoV-2 Nsp13 helicase functions with broad anti-hCoV activity and combination effects with approved antivirals.

Abstract

The SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase essential for viral replication and immune evasion. Its conservation across coronaviruses makes it an attractive target for the development of broad-spectrum antiviral agents. Hence, we rationally designed and synthesised 42 novel 2-iminothiazolidin-4-one derivatives as potential inhibitors. Several compounds displayed low-micromolar IC50 values against both nsp13 NTPase and unwinding activities. Among these, 3i emerged as the lead compound, combining enzymatic inhibition (IC50unwinding 2.65 ± 0.51 μM and IC50 NTPase 8.52 ± 0.68 μM) with a favourable antiviral activity/cytotoxicity profile in two complementary cell models of SARS-CoV-2 infection (EC50 23.80 ± 1.80 μM in VeroE6-GFP, SI 18.15; EC50 6.7 ± 0.86 μM in Calu-3 cells, a human lung epithelial model). 3i also showed antiviral activity against hCoV-229E and MERS-CoV, with EC50 values of 5.7 ± 0.1 and 4.6 ± 1.1 μM, respectively, supporting activity across human coronaviruses. The compound further demonstrated no marked hepatocellular cytotoxicity under the tested conditions in HepG2 cells, negligible inhibition of human helicases DDX3X and DDX5. Experimental biomimetic HPLC analyses provided an initial assessment of its physicochemical and distribution properties. Binding affinity evaluation by SPR revealed a KD value of 6.20 μM, while enzyme kinetic studies indicated mixed-type NTPase inhibition. Exploratory docking and molecular dynamics analyses predicted possible binding regions that may account for the simultaneous modulation of the two nsp13 enzymatic functions. Finally, 3i showed a favourable antiviral combination profile with Remdesivir or Nirmatrelvir, mirroring established combination strategies in antiviral therapy.