Seleno-Functionalization of Quercetin Improves the Non-Covalent Inhibition of Mpro and Its Antiviral Activity in Cells against SARS-CoV-2

Abstract

The development of new antiviral drugs against SARS-CoV-2 is a valuable long-term strategy to protect the global population from the COVID-19 pandemic complementary to the vaccination. Considering this, the viral main protease (Mpro) is among the most promising molecular targets in light of its importance during the viral replication cycle. The natural flavonoid quercetin 1 has been recently reported to be a potent Mpro inhibitor in vitro, and we explored the effect produced by the introduction of organoselenium functionalities in this scaffold. In particular, we report here a new synthetic method to prepare previously inaccessible C-8 seleno-quercetin derivatives. By screening a small library of flavonols and flavone derivatives, we observed that some compounds inhibit the protease activity in vitro. For the first time, we demonstrate that quercetin (1) and 8-(p-tolylselenyl)quercetin (2d) block SARS-CoV-2 replication in infected cells at non-toxic concentrations, with an IC50 of 192 μM and 8 μM, respectively. Based on docking experiments driven by experimental evidence, we propose a non-covalent mechanism for Mpro inhibition in which a hydrogen bond between the selenium atom and Gln189 residue in the catalytic pocket could explain the higher Mpro activity of 2d and, as a result, its better antiviral profile.

Sub-Micromolar Inhibition of SARS-CoV-2 3CLpro by Natural Compounds

“…Figure 2 shows the data obtained for eugenol and estragole, by using the same experimental setup and conditions already reported [9,10,18]. The catalytic activity of 3CLpro was monitored in vitro by using a Förster resonance energy transfer (FRET) continuous assay, in the presence of the substrate (Dabcyl)KTSAVLQSGFRKME(Edans)- These terpenes are volatile substances commonly found in spices and aromatic herbs including clove, basil, cinnamon, pepper, fennel, and anise [20], and are generally considered safe for human use [21].…”

“…Figure 2 shows the data obtained for eugenol and estragole, by using the same experimental setup and conditions already reported [ 9 , 10 , 18 ]. The catalytic activity of 3CLpro was monitored in vitro by using a Förster resonance energy transfer (FRET) continuous assay, in the presence of the substrate (Dabcyl)KTSAVLQSGFRKME(Edans)-NH2.…”

“…This observation, combined with 3CLpro engagement demonstrated by the CD and fluorescence data (Figure 3), suggests that eugenol exerts its inhibition activity as a direct action on the protein catalytic site and, in turn, it has an allosteric effect that promotes 3CLpro oligomerization. In the absence of structural data to confirm the exact location of the ligand bound to 3CLpro, we used computational techniques to gain insight into the anchoring mode, as previously described [9,10,18]. The results of docking simulations performed with Auto-Dock Vina [25] predicted the binding into the 3CLpro catalytic site with affinity at least in the micromolar range (binding energy < −5.0 kcal/mol).…”

“…This observation could explain why neither eugenol nor estragole were reported in previous large-scale computational screening of flavonoids [27,28]-at variance with other natural compounds later confirmed to inhibit 3CLpro. In the absence of structural data to confirm the exact location of the ligand bound to 3CLpro, we used computational techniques to gain insight into the anchoring mode, as previously described [9,10,18]. The results of docking simulations performed with AutoDock Vina [25] predicted the binding into the 3CLpro catalytic site with affinity at least in the micromolar range (binding energy < −5.0 kcal/mol).…”

“…Thus, their common scaffold can be considered promising for further optimization. A seleno-functionalized quercetin analog was later proved to inhibit SARS-CoV-2CoV-2 replication in infected cells at non-toxic concentration, with an IC 50 value of 8 μM [ 18 ].…”