Led by Prof. Amadeu Llebaria from Barcelona, Spain, this project aimed to explore the binding properties of two families of antiviral compounds identified as potential inhibitors of SARS-CoV-2 and related coronaviruses. The research was supported by ISIDORe and facilitated by collaboration with the Goethe-Universität Frankfurt facilities in Germany. The project utilized NMR (Nuclear Magnetic Resonance) to investigate how these compounds interact with critical viral components, particularly the main protease (3CLpro) and viral RNA.
The project originated from the synthesis of more than 200 structurally related compounds designed to target the viral protease and inhibit viral replication. Initial assays revealed two promising families of compounds: Family 1, which showed activity in both enzymatic and cellular assays, and Family 2, which displayed activity primarily in cellular assays, suggesting a different mechanism of action. The goal of this project was to gain insights into the structure-activity relationship (SAR) of Family 1 with 3CLpro and to explore whether Family 2 compounds interact with viral RNA.
NMR experiments were performed on 21 compounds, with a focus on identifying binding interactions with SARS-CoV-2’s nsp5 (3CLpro) and RNA construct 5’SL1234. However, 9 compounds were discarded due to solubility issues. Binding was assessed using several NMR-based methods, including waterLOGSY and saturation transfer difference (STD) experiments, allowing the identification of ligands that bound to the viral protease and RNA.
The results indicated that three compounds from Family 1 interacted with nsp5, with their binding epitopes located near the catalytic dyad (H41 and C145) of the protease. For Family 2, six compounds were found to bind to the SARS-CoV-2 RNA construct 5’SL1234, confirming a potential RNA-targeting mechanism of action for this group of antivirals.
These findings open the door to optimizing these compounds through hit-to-lead projects, with the possibility of developing a second generation of inhibitors. The project also suggests the potential for synergistic antiviral effects by targeting both the viral protease and RNA simultaneously, which could lead to a novel class of antivirals with enhanced efficacy.