Potential In Vitro Inhibition of Selected Plant Extracts against SARS-CoV-2 Chymotripsin-Like Protease (3CLPro) Activity

Abstract

Antiviral treatments inhibiting Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication may represent a strategy complementary to vaccination to fight the ongoing Coronavirus disease 19 (COVID-19) pandemic. Molecules or extracts inhibiting the SARS-CoV-2 chymotripsin-like protease (3CL^Pro) could contribute to reducing or suppressing SARS-CoV-2 replication. Using a targeted approach, we identified 17 plant products that are included in current and traditional cuisines as promising inhibitors of SARS-CoV-2 3CL^Pro activity. Methanolic extracts were evaluated in vitro for inhibition of SARS-CoV-2 3CL^Pro activity using a quenched fluorescence resonance energy transfer (FRET) assay. Extracts from turmeric (Curcuma longa) rhizomes, mustard (Brassica nigra) seeds, and wall rocket (Diplotaxis erucoides subsp. erucoides) at 500 µg mL^-1 displayed significant inhibition of the 3CL^Pro activity, resulting in residual protease activities of 0.0%, 9.4%, and 14.9%, respectively. Using different extract concentrations, an IC₅₀ value of 15.74 µg mL^-1 was calculated for turmeric extract. Commercial curcumin inhibited the 3CL^Pro activity, but did not fully account for the inhibitory effect of turmeric rhizomes extracts, suggesting that other components of the turmeric extract must also play a main role in inhibiting the 3CL^Pro activity. Sinigrin, a major glucosinolate present in mustard seeds and wall rocket, did not have relevant 3CL^Pro inhibitory activity; however, its hydrolysis product allyl isothiocyanate had an IC₅₀ value of 41.43 µg mL^-1. The current study identifies plant extracts and molecules that can be of interest in the search for treatments against COVID-19, acting as a basis for future chemical, in vivo, and clinical trials.

Keywords: COVID-19; FRET assay; SARS-CoV-2 3CLPro; allyl isothiocyanate; curcumin; mustard seeds; natural compounds; plant extracts; turmeric; wall rocket.

Figure 1

Mean values and standard errors (SEs) for the residual activity of SARS-CoV-2 3CL^Pro in the presence of the fifteen plant-based extracts at a concentration of 500 µg mL⁻¹ and the inhibitor control GC376 at a concentration of 50 µM (n = 4). Extracts included were obtained from citrus fruit peels (blue), seasoning and aromatic herbs (green), bulbs and rhizomes (purple; includes turmeric), a succulent plant (red), and cruciferous herbs and condiments (yellow). Both the turmeric extract and the inhibitor GC376 produced a complete inhibition of the 3CL^Pro. Different letters indicate significant differences in the 3CL^Pro residual activity according to a Student–Newman–Keuls test at p < 0.05.

Figure 2

(a) Dose–response curve for the methanolic extract of turmeric powder. Each point represents the average value and SE for the residual activity of the 3CL^Pro (%) at extract concentrations between 5.0 and 500 µg mL⁻¹ (n = 4). The 100% of residual activity considers the activity of the 3CL^Pro when no inhibitor is added. (b) Mean residual activity and SE of 3CL^Pro (%) determined after the co-incubation with five different concentrations of commercial curcumin (2.5–75 µg mL⁻¹) (n = 4). Different letters indicate significant differences in the 3CL^Pro residual activity according to a Student–Newman–Keuls test at p < 0.05.

Figure 3

(a) Dose–response curve for the methanolic extracts of mustard seeds (blue circles) and wall rocket leaves (orange squares). Each point represents the average value and SE for the residual activity of the 3CL^Pro (%) at extract concentrations between 5.0 and 500 µg mL⁻¹ (n = 4). The 100% of residual activity is the activity of the 3CL^Pro when no inhibitor is added. (b) Mean residual activity and SE of 3CL^Pro (%) determined after the co-incubation with 5.0–500 µg mL⁻¹ of sinigrin (grey) and allyl isothiocyanate (black) standards (n = 4). Different letters indicate significant differences in the 3CLPro residual activity for sinigrin or allyl isothiocyanate treatments according to two separate Student–Newman–Keuls tests at p < 0.05.