Commercially Available Flavonols Are Better SARS-CoV-2 Inhibitors than Isoflavone and Flavones

Abstract

Despite the fast development of vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still circulating and generating variants of concern (VoC) that escape the humoral immune response. In this context, the search for anti-SARS-CoV-2 compounds is still essential. A class of natural polyphenols known as flavonoids, frequently available in fruits and vegetables, is widely explored in the treatment of different diseases and used as a scaffold for the design of novel drugs. Therefore, herein we evaluate seven flavonoids divided into three subclasses, isoflavone (genistein), flavone (apigenin and luteolin) and flavonol (fisetin, kaempferol, myricetin, and quercetin), for COVID-19 treatment using cell-based assays and in silico calculations validated with experimental enzymatic data. The flavonols were better SARS-CoV-2 inhibitors than isoflavone and flavones. The increasing number of hydroxyl groups in ring B of the flavonols kaempferol, quercetin, and myricetin decreased the 50% effective concentration (EC₅₀) value due to their impact on the orientation of the compounds inside the target. Myricetin and fisetin appear to be preferred candidates; they are both anti-inflammatory (decreasing TNF-α levels) and inhibit SARS-CoV-2 mainly by targeting the processability of the main protease (M^pro) in a non-competitive manner, with a potency comparable to the repurposed drug atazanavir. However, fisetin and myricetin might also be considered hits that are amenable to synthetic modification to improve their anti-SARS-CoV-2 profile by inhibiting not only M^pro, but also the 3'-5' exonuclease (ExoN).

Keywords: COVID-19; SARS-CoV-2; calu-3 cells; exonuclease; flavonoids; molecular docking; natural product; protease.

Figure 1

The chemical structures of the flavonoids evaluated in this work.

Figure 2

Antiviral activity of flavonoids and RDV (positive control). (A) SARS-CoV-2 production and (B) inhibition of SARS-CoV-2 replication in Calu-3 cells (densities of 2.0 × 10⁵ cells/well) infected with SARS-CoV-2 B.1 lineage at a MOI of 0.1.

Figure 3

The anti-inflammatory profile of flavonoids was assessed using the (A) IL-6 and (B) TNF-α levels from uninfected Calu-3 cells supernatant (MOCK), SARS-CoV-2 infected cells without treatment (NIL), and infected and flavonoid treated cells with (10 µM). Blue, green, and red bars correspond to isoflavone, flavones, and flavonols, respectively. * p < 0.05.

Figure 4

The electrostatic potential map (−68.065 and +68.065 a.u, in red and blue, respectively) of the best docking poses for the flavonoids (A) isoflavone, (B) flavones, and (C) flavonols in the SARS-CoV-2 nsp14 ExoN active site in the presence of one and two Mg(II) ions (left and right, respectively). The catalytic amino acid residues, genistein, apigenin, luteolin, fisetin, kaempferol, myricetin, and quercetin are in stick representation in cyan, violet, pink, brown, gray, light green, purple, and marine, respectively. Mg(II) and Zn(II) ions are represented as green and indigo blue spheres, respectively. Hydrogen, oxygen, and nitrogen are shown in white, red, and dark blue, respectively.

Figure 5

The main amino acid residues from SARS-CoV-2 nsp14 ExoN that interact with (A) fisetin and (B) myricetin in the presence of one Mg(II) ion, and (C) fisetin and (D) myricetin in the presence of two Mg(II) ions. The interactive and catalytic amino acid residues are in beige and cyan, respectively; fisetin and myricetin are in gray and purple, respectively. Mg(II) ions are represented as green spheres. Hydrogen, oxygen, and nitrogen are shown in white, red, and dark blue, respectively.

Figure 6

Inhibition of SARS-CoV-2 ExoN activity by fisetin. A mixture of 500 nM RNA (sequence shown at the top of the figure) and 50 nM SARS-CoV-2 pre-assembled exonuclease complex (nsp14/nsp10) was incubated in buffer solution at 37 °C for 15 min (B) in the absence and (C,D) presence of 50 μM and 150 μM fisetin. The (A) RNA and the (B–D) products of the ExoN reaction were analyzed by MALDI-TOF MS. The signal intensity was normalized to the highest peak. The peak at 8164 Da corresponds to the intact RNA (8157 Da expected).

Figure 7

The 3D-representation of the best docking pose and the corresponding zoom representation for the flavonoids (A) isoflavone, (B) flavones, and (C) flavonols in the SARS-CoV-2 M^pro active site in the presence of peptide substrate (CAS number 730985-86-1). Catalytic amino acid residues genistein, apigenin, luteolin, fisetin, kaempferol, myricetin, and quercetin are in stick representation in cyan, violet, pink, brown, gray, light green, purple, and marine, respectively. Catalytic water (H₂O_cat) is shown in spherical configuration. Hydrogen, oxygen, and nitrogen are shown in white, red, and dark blue, respectively.

Figure 8

(A) Inhibition percentage of flavonoids (10 μM) to 88.8 nM M^pro. Blue, green, and red bars correspond to isoflavone, flavones, and flavonols, respectively. (B) The enzymatic inhibition profile for fisetin, myricetin, and GC376 (positive control) for 88.8 nM M^pro in PBS. (C) Michaelis–Menten enzymatic mechanism for M^pro in the absence and presence of a fixed fisetin or myricetin concentration (2.5 µM) at different substrate concentrations.