A new study by Italian researchers have found that NAC or N-Acetylcysteine is not only an effective antiviral agent against all of the SARS-CoV-2 variants but also has immunomodulatory properties that can help prevent disease severity.
The study findings were published on a preprint server but is currently being peer reviewed.
As obligate intracellular parasites, viruses tightly rely on their host cells: they have evolved to exploit cells for their own purposes by hijacking cellular pathways and to evade the innate immune response by modulating host factors and signalling pathways.
RNA viruses, such as SARS-CoV-2, even more heavily rely on the host cell(57). However, current therapeutic interventions against COVID-19 are solely targeted against viral proteins, promoting the emergence of variants escaping vaccine-induced immunity or resistance to antiviral drugs.
The objective of this study was understanding whether targeting host proteins might be an effective and safe strategy against COVID-19, as host genes are not under selective pressure. We started by asking whether different SARS-CoV-2 variants elicit similar cellular responses upon infection.
The three major SARS-CoV-2 variants of concern that we used in our study exhibited varying replication patterns in human lung cells, with Wuhan producing fewer transcripts and higher titres of infective viral progenies when compared to D614G and Alpha.
We ascribed this diverse behaviour to the reported mutations in the Spike protein, which would result in modulated virus-receptor binding affinity and consequent viral entry(58). In addition, both D614G and Alpha harbour mutations and deletions in the open-reading frame 8 (ORF8) (Supplementary Table 1), which inhibits the cell interferon-mediated immune response(18, 59, 60), possibly explaining the enhanced RNA transcription of these two variants(61, 62).
However, analyses of the host transcriptome revealed a qualitatively highly similar transcriptional response among the 3 variants, with differences in just kinetics and magnitude, overall indicating that different variants induce similar cellular responses upon infection.
We then applied a genome-wide CRISPR knockout approach to gather deep insights into the host genes exploited by different variants, asking whether some genes are specifically required for the infection of one or more variants. This kind of approach has been successfully developed to identify the host factors exploited by other viruses(63–67) and by SARS-CoV-2 itself(4–8, 68, 69).
However, the analysis of available data of previous SARS-CoV-2 CRISPR knockout screenings does not allow us to draw conclusions about whether different variants exploit different host factors, because different studies used different combinations of variants, cell lines and CRISPR libraries.
For these reasons, we performed a genetic screening directly comparing 3 variants under identical conditions and looked for the host factors that are conservatively exploited by all of them and, vice versa, those that are required by specific variants. The rationale of our approach is twofold:
i) if a host factor is shared by all variants, it more likely belongs to a “core” of host factors essential for the viral infection and
ii) shared host factors are more likely to be required by new variants of SARS-CoV-2 that will emerge in the future and thus might serve as a better and omni-comprehensive therapeutic target.
By using conditions ensuring high coverage and stringency, we retrieved 525 genes, the knockout of which allowed cell survival upon infection; 93.3% were shared by at least 2 out of 3 variants. Very satisfactorily, all candidates selected by the CRISPR knock-out screening were also confirmed by transient silencing of host genes. Importantly, we failed to identify a single candidate acting specifically on only 1 variant. We conclude that the host factors exploited during infection are highly shared among different SARS-CoV-2 variants.
We believe that the knowledge acquired in this study will be instrumental to develop host-directed therapies to control SARS-CoV-2 infection. Due to their reliance on host cell components, these have reduced likelihood to develop resistance. To further assess the soundness of our hits and provide ready-to-trial drugs able to stop viral infection/replication of present and forthcoming variants, we screened a set of FDA-approved drugs against unrelated diseases, and chemical compounds reported to hamper the main common viral host factor candidates (SLC7A11, RIPK4 and MASTL).
The five tested compounds displayed potent antiviral activity not only against the three tested SARS-CoV-2 variants, but also against the Delta variant, which appeared in late 2021 and has been so far the last variants that caused worrisome rates of hospitalisation of infected patients of all ages, regardless of their vaccination status, and was associated with high mortality rate(70, 71).
The successful antiviral activity of the tested compounds further reinforces the strength of our screening, and points out that the selected hits are crucial host factors for both the early and latest variants.
The mechanism of action of one of the most promising tested compounds, IKE, was investigated to further validate its target, SLC7A11, against SARS-CoV-2. The central role of SLC7A11 in the maintenance of ROS intracellular homeostasis and its relevance as host factor in different human viral infections have been previously reported(37, 72–75).
IKE was proposed to neutralise SLC7A11-mediated cystine uptake and ROS modulation(76). While increased intracellular ROS levels trigger innate immunity-mediated antiviral mechanisms, counterintuitively, viral infections stimulate ROS production and viruses thrive in increased ROS levels(55, 77).
Indeed, our gene expression analysis suggests reduced oxidative phosphorylation within infected cells, possibly as an attempt the cells make to lower ROS and create a hostile environment for viral replication. We showed that SARS-CoV-2 stimulates ROS production during the early infective stages in human bronchial cells. Reduction of ROS levels, by extended IKE administration, glutathione or NAC treatment, impaired SARS-CoV-2 viral cycle.
The effect of NAC treatment in COVID-19-affected patients has been investigated in several retrospective studies leading to suggestive, albeit not definitive, results (78–80). The mechanistic explanation was that the antioxidant, anti-inflammatory and anti-thrombotic effects of NAC counteracted viral pneumonia; however results from ongoing randomised controlled trials are required to draw accurate conclusions (79). In the meanwhile, our results show a direct antiviral effect of NAC on lung epithelial cells, in addition to its immunomodulatory effects. We thus strongly encourage and support NAC, and other antioxidant drugs, use as a safe and accessible anti-SARS-CoV-2 therapy, against current and future variants.