Effects of Sulforaphane on SARS‑CoV‑2 infection and NF‑κB dependent expression of genes involved in the COVID‑19 'cytokine storm'.

Since its spread at the beginning of 2020, the coronavirus disease 2019 (COVID‑19) pandemic represents one of the major health problems. Despite the approval, testing, and worldwide distribution of anti‑severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) vaccines, the development of specific antiviral agents targeting the SARS‑CoV‑2 life cycle with high efficiency, and/or interfering with the associated 'cytokine storm', is highly required. A recent study, conducted by the authors' group indicated that sulforaphane (SFN) inhibits the expression of IL‑6 and IL‑8 genes induced by the treatment of IB3‑1 bronchial cells with a recombinant spike protein of SARS‑CoV‑2. In the present study, the ability of SFN to inhibit SARS‑CoV‑2 replication and the expression of pro‑inflammatory genes encoding proteins of the COVID‑19 'cytokine storm' was evaluated. SARS‑CoV‑2 replication was assessed in bronchial epithelial Calu‑3 cells. Moreover, SARS‑CoV‑2 replication and expression of pro‑inflammatory genes was evaluated by reverse transcription quantitative droplet digital PCR. The effects on the expression levels of NF‑κB were assessed by western blotting. Molecular dynamics simulations of NF‑kB/SFN interactions were conducted with Gromacs 2021.1 software under the Martini 2 CG force field. Computational studies indicated that i) SFN was stably bound with the NF‑κB monomer; ii) a ternary NF‑kB/SFN/DNA complex was formed; iii) the SFN interacted with both the protein and the nucleic acid molecules modifying the binding mode of the latter, and impairing the full interaction between the NF‑κB protein and the DNA molecule. This finally stabilized the inactive complex. Molecular studies demonstrated that SFN i) inhibits the SARS‑CoV‑2 replication in infected Calu‑3 cells, decreasing the production of the N‑protein coding RNA sequences, ii) decreased NF‑κB content in SARS‑CoV‑2 infected cells and inhibited the expression of NF‑kB‑dependent IL‑1β and IL‑8 gene expression. The data obtained in the present study demonstrated inhibitory effects of SFN on the SARS‑CoV‑2 life cycle and on the expression levels of the pro‑inflammatory genes, sustaining the possible use of SFN in the management of patients with COVID‑19.