In Vitro Antiviral Activity of Nordihydroguaiaretic Acid against SARS-CoV-2

Abstract

The coronavirus infectious disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has been spreading rapidly worldwide, creating a pandemic. This article describes the evaluation of the antiviral activity of nordihydroguaiaretic acid (NDGA), a molecule found in Creosote bush (Larrea tridentata) leaves, against SARS-CoV-2 in vitro. A 35 µM concentration of NDGA was not toxic to Vero cells and exhibited a remarkable inhibitory effect on the SARS-CoV-2 cytopathic effect, viral plaque formation, RNA replication, and expression of the SARS-CoV-2 spike glycoprotein. The 50% effective concentration for NDGA was as low as 16.97 µM. Our results show that NDGA could be a promising therapeutic candidate against SARS-CoV-2.

Keywords: Larrea tridentata; NDGA; SARS-CoV-2 antivirals.

Figure 1

Cytotoxicity of nordihydroguaiaretic acid in Vero cells. (A) Chemical structure of NDGA; (B) Cell viability of Vero cells treated with NDGA (≥90% purity) after 48 h treatment, using the MTT assay. Each value represents the mean of two experiments with four replicates ± standard deviation (SD). CC₅₀ value was determined using non-linear regression with GraphPad Prism 8, and the blue line represents the tendency line of analysis.

Figure 2

Inverted microscopic representative photographs of cell cultures at 96 h post-infection. Vero CCL-81 cells were infected with 100 TCID₅₀/mL of SARS-CoV-2 and treated with two NDGA concentrations. (A) Negative control (untreated Vero cells); (B) SARS-CoV-2 positive control presenting CPE (black arrow: cell rounding; yellow arrow: detachment); (C) Infected Vero cells treated with NDGA 35 µM; (D) Infected Vero cells treated with NDGA 50 µM. (E) Dose–response curve analyses were performed against 100 TCID₅₀ viral concentrations. Cytopathic effect reduction was expressed as the percent protection from CPE (no CPE appearance in replicates n = 8) in two experiments. The EC50 value was determined using non-linear regression with GraphPad Prism 8, and the blue line represents the tendency line of analysis.

Figure 3

Antiviral activity of NDGA against SARS-CoV-2 by viral plaque reduction assay at 96 h post-infection. (A) Simultaneous infection assay with 35 µM NDGA, plus 100 TCID₅₀/mL of SARS-CoV-2; (B) Diagram of the time of drug assay; (C) Time of addition analysis of NDGA against 250 TCID₅₀/mL of SARS-CoV-2 infection. The experiments were carried out in quadruplicate, and the percentage of reduction is represented as the mean ± S.D. * p ≤ 0.05, as compared with control; (D) Western blot analysis. SARS-CoV-2 spike protein was detected in the positive control but not with 35 µM of NDGA treatment. Bio-Rad Precision Plus Standards were loaded in line 1.

Figure 4

Quantification of viral genome replication in cell culture. Vero cells were treated with NDGA 35 µM and infected simultaneously with 100 TCID₅₀/mL of each SARS-CoV-2 variant, for 96 h. as described in Section 2.6. All assays were performed in triplicate. Viral RNA from positive control (untreated) supernatants or treated cultures were measured by real-time RT-qPCR. The student t-test was performed, and data is reported as mean values ± SD (**** p < 0.001).