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 (Mpro) 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 Mpro, but also the 3′–5′ exonuclease (ExoN).

SARS-CoV-2 Mpro Inhibitors: Achieved Diversity, Developing Resistance and Future Strategies

“…In vitro screening confirmed sub-micromolar inhibition (IC50 = 0.2-0.6 µM), and in the solid-state structure, the covalent linkage to C6 of the pyrogallol unit is obvious (PDB 7b3e). Alternatively, a noncompetitive inhibition mechanism is proposed in the literature [21]. Myricetin's carbonyl-rich structure enables stabilization by hydrogen-bonding, which is moderated through active site water molecules [22].…”

“…Covalent bond formation via pyrogallol-C6 can be explained by a preceding oxidation of pyrogallol to o-quinone, enabling attack of the nucleophilic Cys145 thiolate. Myricetin was further shown to block viral infection in vitro (EC50 = 8 μM (VeroE6, [22]); 0.9 μM (Calu-3), [21]) and reduce lung inflammation in a mouse model of bleomycin-induced pulmonary inflammation, making it potentially useful for the symptomatic treatment of COVID-19 [23]. To increase potency, myricetin analogs were designed that included compound 3 with a diphenyl phosphate to the 7-OH of myricetin, resulting in a stronger antiviral effect (Figure 3; EC50 = 3.15 μM) [22].…”

“…Presumably due to a limited cell membrane permeability, MI-23 and other inhibitors of this series had reduced activity in the cell protection assay (EC50 = 5.6 µM), incentivizing the authors to select further structurally modified P3 analogs for pharmacological testing. The analog MI-30 (21) with a (2,4-dichlorophenoxy)acetyl group in P3 had a persistent low IC50 of 0.017 µM, good in vitro efficacy (EC50 = 0.54 µM), no common cell toxicology, and protected transgenic mice that expressed hACE2 against SARS-CoV-2 infection. In an SD rat model, MI-30 showed good in vivo stability (t0.5 = 2.35 h), high plasma concentrations (Cmax = 23582 ng/mL), and sufficient clearance (CL = 17.1 mL/min/kg).…”

“…Another set of studies proved calpain inhibitors II and XII as potent SARS-CoV-2 M pro binders (IC50 = 0.97 and 0.45 μM, respectively) with a micromolar antiviral effect [39,58,59]. [41,42], GC376 and GC373 [43], 16 and 17 [48], UAWJ247 [49], NK01-63 [50], 18 [51], 19 and 20 [52], MI-23 and MI-30 (21) [53], MPI8 [54], as well as MG-132 [56]. Inhibitory (IC50) and antiviral data (EC50) are given below each structure.…”

Agathisflavone, a natural biflavonoid that inhibits SARS-CoV-2 replication by targeting its proteases

“…In this context, polyphenolic metabolites, such as flavonoids, represent a class of molecules that includes the largest source of substances with antiviral activity [19] . Indeed, numerous flavonoids, e.g., fisetin, myricetin, quercetin, and kaempferol, have been described with antiviral activity, targeting proteases (M pro or PL pro ) of SARS-CoV, MERS-CoV, and SARS-CoV-2 [19] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] . On the other hand, recent reports indicated that natural dimers of flavonoids, known as biflavonoids, frequently reported in plants used in traditional and modern medicine, have attracted more attention than flavonoids due to their highest anti-inflammatory, antioxidant, antibacterial, antidiabetic, antitumor, and antiviral (including anti-SARS-CoV and anti-SARS-CoV-2) properties [31] , [32] , [33] , [34] .…”