Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.
The coronavirus disease 2019 (COVID-19) pandemic has resulted in substantial global morbidity and mortality. While an unprecedented effort has led to the development of highly effective vaccines, many people remain vulnerable to developing severe disease due to inadequate accessibility or unwillingness to be vaccinated, as well as poor immune responses in certain populations. Other therapeutic approaches have also been developed for COVID-19, including early treatments with monoclonal antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)1, convalescent plasma2,3, and antivirals4. Immunomodulators have also been utilized to modify disease and prevent mortality5. Early intervention after symptom onset has been shown to be most effective in preventing severe disease and hospitalizations6,7. Therefore, the ideal therapy should be one that is readily available and easily administered to patients. Among the direct-acting antivirals, molnupiravir and ritonavir-boosted nirmatrelvir (Paxlovid) are the only oral agents currently authorized by the United States Food and Drug Administration for the treatment of patients with COVID-197,8,9. Additional oral antiviral therapeutics are urgently needed to prevent more severe disease, hospitalization, and death.
The multi-functional phytochemical sulforaphane (SFN) is the isothiocyanate derived from enzymatic hydrolysis of its precursor glucoraphanin, a glucosinolate found in high concentrations in broccoli (Brassica oleracea italica) and other cruciferous vegetables. SFN is a potent naturally occurring activator of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), with well-documented antioxidant and anti-inflammatory effects10,11,12. Treatment with SFN increased phagocytic activity of alveolar macrophages13and reduced lung injury in animal models of acute respiratory distress syndrome (ARDS)14. SFN also decreased the levels of IL-6 and viral load in human subjects infected with live attenuated influenza virus15,16. Numerous clinical trials utilizing SFN have demonstrated favorable pharmacokinetics after oral dosing and documented excellent tolerability and safety10,17,18,19.
SFN was identified after an exploratory screening of readily available drugs and compounds for efficacy against human coronaviruses. Initial testing was performed in vitro using seasonal coronavirus HCoV-OC43. Subsequently, drugs that exhibited at least moderate activity against HCoV-OC43 were tested in vitro against SARS-CoV-2. We report here that SFN inhibits in vitro HCoV-OC43 and SARS-CoV-2 infections of mammalian cells and appears to have a synergistic interaction with remdesivir. In addition, SFN reduces viral load and pulmonary pathology in a mouse model of SARS-CoV-2 infection
The ongoing SARS-CoV-2 pandemic has created the immediate need for effective therapeutics that can be rapidly translated to clinical use. Despite the introduction of vaccines, effective antiviral agents are still necessary, particularly considering the potential effects of viral variants26. New oral antivirals targeting viral enzymes (e.g., molnupiravir and Paxlovid) have recently been approved or are in the process of review for emergency use approval by regulatory agencies, with many more currently under development7,8,27. However, this approach can be affected by the emergence of viral variants that change the affinity of the drug to the viral protein28. An alternative approach is to target host mechanisms required by the virus to infect cells and replicate29. Host-directed therapy is advantageous as it allows preexisting drugs to be repurposed, may provide broad-spectrum inhibition against multiple viruses, and is generally thought to be more refractory to viral escape mutations30,31.
Following exploratory experiments using the in vitro CPE inhibition assay, SFN was identified as a promising candidate to target the host cellular response, given that it is orally bioavailable, commercially available at low cost, and has limited side effects18,32. We observed that SFN has dual antiviral and anti-inflammatory properties against coronaviruses. We determined that SFN has potent antiviral activity against HCoV-OC43 and multiple strains of SARS-CoV-2, including Delta and Omicron, with limited toxicity in cell culture. The similar results observed between the coronaviruses evaluated suggest that SFN could have broad activity against coronaviruses, a feature that may prove invaluable as new strains of pathogenic coronaviruses enter the human population. Moreover, synergistic antiviral activity was observed in vitro between SFN and remdesivir against both types of coronaviruses tested; comparable synergism in vivo would be advantageous in clinical scenarios where remdesivir is currently being used. We demonstrated in vivo efficacy of prophylactic SFN treatment using the K18-hACE2 mouse model of SARS-CoV-2 infection22. Prophylactic SFN-treatment in animals reduced viral replication in the lungs by 1.5 orders of magnitude, similar to that reported for remdesivir in the same mouse model33. By comparison, BALB/c mice infected with mouse-adapted SARS-CoV-2 had a 1.4 log10 reduction in viral titers when treated with 300 mg/kg of nirmatrelvir 4 h after infection8. As expected, SFN treatment also modulated the inflammatory response in SARS-CoV-2-infected mice, leading to decreased lung injury.
The pathogenesis of many viral infections is associated with increased production of reactive oxygen species (ROS), which leads to cell death34,35,36. Conversely, SFN increases antioxidant, anti-inflammatory, and antiviral defenses through multiple mechanisms1,7, including the activation of the cap’n’collar transcription factor NRF237. Under normal conditions, NRF2 remains in an inactive state by association with its inhibitor protein Kelch-like ECH-associated protein 1 (KEAP1)38. In response to oxidative stress, KEAP1 is inactivated, and NRF2 is released to induce NRF2-responsive genes that subsequently protect against stress-induced cell death39. SFN has been extensively studied in humans for its anti-cancer properties, has been shown to activate the NRF2 pathway in upper airways40, and improves the phagocytic ability of alveolar macrophages13. The dual antiviral and anti-inflammatory properties of SFN have also been previously described for other viral infections. In vitro antiviral activity has been reported against influenza virus41, and SFN treatment significantly limited lung viral replication and virus-induced inflammation in respiratory syncytial virus-infected mice42.
SFN also inhibits inflammation through NRF-2 independent pathways, such as reducing the proinflammatory nuclear factor kappa B (NF-κB)43. NF-κB activation has been described as a key component of the inflammatory response to multiple viral infections, including COVID-1944. There are also other pathways affected by SFN (e.g., STING, STAT3, macrophage migration inhibitory factor) that could play a role in its antiviral response to coronaviruses45. While NRF-2 activation and enhanced transcription of its target genes usually require longer periods of time, we observed potent antiviral activity in cells that had been treated with SFN for only 1–2 h. In NRF2-KD cells infected with SARS-CoV-2, SFN treatment was still able to significantly reduce the viral load. Therefore, it is possible that the antiviral effect of SFN is NRF-2 independent while the anti-inflammatory effects are mediated primarily by NRF2. Further studies are needed to determine the contribution of each of the different cellular pathways to the antiviral activity of SFN.
As a potent NRF2 activator, SFN can modulate the host’s immune response while also providing direct, NRF2-independent antiviral effects. Targeting the NRF2 pathway has been considered a promising approach to develop therapeutics for COVID-19 for multiple reasons46,47,48. NRF2 deficiency is known to upregulate the angiotensin-converting enzyme 2 (ACE2), the primary mechanism of cell entry for SARS-CoV-2. The NRF2 activator oltipraz reduces ACE2 levels, suggesting that NRF2 activation might reduce the availability of ACE2 for SARS-CoV-2 entry into the cell49. Increased NRF2 activity also reportedly inhibits IL-6 and IL-1β gene expression50, two cytokines known to play key roles in promoting the hyperactive immune response in severely ill COVID-19 patients51. Conversely, NRF2 activity is dysregulated in disease states that have been associated with increased severity of COVID-19 (e.g., diabetes)52. Further, NRF2 activity declines in older patients who are more susceptible to severe COVID-1953. Recent reports suggest that NRF2-dependent genes are suppressed in SARS-CoV-2 infected cells and lung biopsies from COVID-19 patients46. Similarly, treatment of cells with NRF2 agonists 4-octyl-itaconate and dimethyl fumarate inhibited replication of SARS-CoV-2 in vitro46.
In contrast to therapeutics that inhibit a single cytokine (e.g., IL-6, IL-1β, etc.)5,54, SFN has important and diverse effects in modulating the lung immune response to SARS-CoV-2 infection. Excessive inflammatory response to SARS-CoV-2 leads to severe disease or death in patients with COVID-1955. Therefore, promoting a balanced and robust antiviral response while modulating excessive innate inflammatory responses could represent a favorable scenario that could reduce viral load while also limiting collateral damage to the infected lung. As has been previously reported, SARS-CoV-2 infection leads to an increase in pulmonary dendritic cells and a reduction in CD4+ T cells in K18-hACE2 mice22. We observed substantial accumulation of immune cells in the lungs of SARS-CoV-2 infected mice, consistent with what has been noted on postmortem analysis of patients with COVID-1922,56, as well as decreased numbers of T cells in the spleen, consistent with human studies where lymphopenia is correlated with severe COVID-1957. SFN treatment had significant effects on multiple immune cell populations in the lungs, with a reduction in monocytes, NK cells, and dendritic cells compared to infected untreated controls. These findings are likely the effect of a combination of the overall reduced inflammation and direct effects of SFN on specific cell populations. For example, NK cells exposed to SFN had increased cell lytic function through dendritic cell-mediated IL-12 production58. We observed decreased recruitment of myeloid cells to the lungs of treated mice and decreased activation profile of local macrophages. The presence of alveolar macrophages with transcriptionally upregulated inflammatory genes and increased secretion of IL-1β have been associated with worse outcomes and increased mortality in patients with ARDS24,59,60,61. Our results show increased IL-1β in alveolar macrophages with SARS-CoV-2 infection, which was abrogated by SFN treatment (P < 0.0001). Mechanistically, the benefits of SFN therapy in our model could be due in part to its modulatory effects on myeloid cells after SARS-CoV-2 infection. SFN treatment led to a reduction in TNF-α in alveolar macrophages and IFN-γ in T cells, both of which are key triggers of cell death and mortality in SARS-CoV-2 infection and cytokine shock syndromes62. Further, SFN was able to reduce but not eliminate T cell activation within the lung. This reduction in T cell activation could be a direct effect in T cells or could operate through downregulation of myeloid costimulatory for T cells such as CD80/CD86. SFN might therefore be able to modulate and dampen immune responses without inhibiting immunity necessary for viral clearance.
While the K18-hACE2 mouse model has been previously used to recapitulate features of COVID-19 in humans22, our study has several limitations. The expression of the hACE2 transgene is non-physiological, resulting in tissue expression levels that are distinct from endogenously expressed ACE2. Sex differences, which are known to occur with SARS-CoV-2 infection, could not be assessed since only male animals were used in these experiments63,64. Finally, the absorption of SFN after oral administration can be modified by the intestinal microbiome10, leading to potentially variable drug exposures between animals.
Our results demonstrate that pharmacologically relevant micromolar concentrations of SFN inhibited viral replication and virus-induced cell death in vitro. Consumption of SFN-rich broccoli sprouts (single oral daily dose equivalent to 200 µmol of SFN) results in a peak plasma concentration (Cmax) of 1.9 µM at 2–3 h65,66, and higher steady-state levels could be achieved by administering the same dose in two divided doses10,65,67. By comparison, SFN inhibited in vitro SARS-CoV-2 replication in human cells with an IC50 of 2.4 µM. It is important to note that the bioavailability of SFN in humans is dependent on many factors including amount consumed, dietary form and preparation technique, and the individual’s gastrointestinal microflora10,68,69. Studies using SFN-rich broccoli sprouts corresponding to 50–400 μmol SFN daily have shown that SFN is well tolerated without clinically significant adverse effects10,32,70,71. Additionally, while SFN is rapidly eliminated from plasma, it reportedly exerts a sustained effect on gene expression72. A daily dose of SFN-rich broccoli sprouts corresponding to 400 μmol (70 mg) of SFN in humans is not equivalent to the 30 mg/kg of SFN used in the current mouse studies. Thus, while our results are promising, additional studies in humans are needed to determine the efficacy of SFN as a therapy for COVID-1968.
In summary, we documented that SFN can inhibit in vitro and in vivo replication of SARS-CoV-2 at pharmacologically and potentially therapeutically achievable concentrations. Further, it can modulate the inflammatory response, thereby decreasing the consequences of infection in mice when administered prior to infection. Given that SFN is orally bioavailable, commercially available, and has limited side effects, our results suggest it could be a promising approach for the prevention and treatment of COVID-19 as well as other coronavirus infections. Further studies are needed to address these possibilities.