The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to circulate around the world, with thousands of new cases being reported daily. The threat of emergence of new variants with greater pathogenicity and the ability to evade the immune response induced by earlier strains of the virus or by vaccination makes it a priority to identify effective and safe antivirals that can prevent or treat infection.
A new paper presents the results of a mouse study using the repurposed antibacterial drug clofoctol against this virus.
The study made use of a drug screen to identify lead compounds from among 2,000 compounds that have been in clinical use or are still in use. This approach is referred to as repurposing and cuts down on the drug development time and costs by examining already approved drugs.
The researchers in this study, published in the journal PLOS Pathogens, made use of their high-content screen (HCS) technique to go through the Apteeus drug library (TEELibrary) of almost 2,000 approved drugs. The objective of this customized cascade of drug evaluation was to find drugs with anti-SARS-CoV-2 activity.
What did the study show?
The study was conducted in Vero cells, first with chloroquine as a dummy. This showed that the drug indeed inhibited the endocytosis of the virus into the cell. However, it failed to affect the alternative TMPRSS2 pathway that uses membrane fusion and thus proved ineffective against the virus. The effect of this drug did allow a benchmark to be established for the assay's dynamic range.
On this basis, they screened the drug library, looking for virus-induced cytopathic effect (CPE) at 72 hours. Of the drugs that inhibited the CPE most potently, many had already been identified in other studies, such as nitazoxanide and amodiaquine. Most of the 57 drugs in this category were basic compounds that accumulate within endosomes, blocking endocytosis by changing endosomal pH.
These were reduced to 21 based on their dose-response curves. Further, Vero cells that express TMPRSS2 were used to ensure that the best of these compounds selected for their inhibitory effect on the virus block both virus entry pathways. Thus, eight compounds were tested on these cells, of which 3 showed dose-dependent antiviral activity.
Of these, clofoctol proved to be a previously unreported compound with high tissue distribution. Interestingly, its lung concentration is double that of plasma.
This drug was sold as Octofene in France until 2005 and continues to be used in Italy as GramPlus to treat Streptococcus pneumoniae and Staphylococcus aureus. Against these pathogens, it inhibited cell wall synthesis and led to increased membrane permeability.
It showed potent inhibition of SARS-CoV-2 replication without cytotoxicity, with the concentration of infectious virions dropping in a dose-dependent manner.
The half-maximal inhibitory concentration (IC50) was 12 μM in Vero-TMPRSS2 cells, confirming its antiviral activity, while in the human-derived Calu-3 cells, it was 8 μM. The mechanism of action was post-inoculation, unlike chloroquine which inhibits viral entry, or the well-known drug remdesivir, which inhibits infection only after viral entry occurs.
Clofoctol inhibited viral translation and replication and remained active when added to the cells 3-4 hours after inoculation. Importantly, it was active against multiple variants, including D614G, Alpha, Beta, and Delta, and against the seasonal endemic human coronavirus 229E.
The specific action of clofoctol was determined to be the translation of a messenger ribonucleic acid (mRNA) that contains the untranslated regions (UTRs) of the virus, thus blocking the genomic and subgenomic transcripts. However, it did not inhibit cellular protein translation.
Further, a mouse study showed a high concentration in the lungs, seven-fold in plasma. When treated with this drug 1 hour and 8 hours from infection for two days, the mice showed weight loss, but the infectious viral load in the lungs decreased by over 1 log on day 2. Male mice have been shown to be more readily infected, but they also showed the same antiviral effect as clofoctol.
The dose-dependent effect of clofoctol led to a reduction in inflammatory gene transcription, such as the interleukins IL-6, IL12p40, tumor necrosis factor (TNF)α, interferons IFNβ, IFNγ, and the interferon-stimulated genes (ISG) Mx1, Ifi44 and ISG15. The viral load continued to be lower for up to four days from inoculation, though the effect waned compared to the second day.
The drug also modulated the reduction in epithelial barrier function gene markers associated with SARS-CoV-2 infection. And finally, the lung inflammation was minimal in treated animals compared to controls.
What are the implications?
The findings of the study show that "at doses that produce lung concentrations close to those observed in human patients treated at the approved dose, clofoctol treatment in mice just after infection lowers SARS-CoV-2 replication and reduces lung pathological features associated with this viral infection."
While clofoctol has been shown to be an inhibitor of viral protein translation, it may have other actions, as well unidentified. The translation inhibition could be a result of activating all three unfolded protein response (UPR) pathways by inducing endoplasmic reticulum stress. Notably, the virus can activate these pathways, but the chemical activation of UPR pathways inhibits coronavirus replication.
The peak concentration of the drug in human lungs is 20 times above the IC50 in Vero cells at less than 2 hours from rectal administration. In the mouse study, the concentration remained steady, well above the IC50 in vitro.
Not only so, but it also reduced the viral load and inflammation in the lungs. This indicates a favorable set of characteristics for its development as a COVID-19 therapeutic.
This is the first time that the anti-inflammatory effects of clofoctol were reported. This drug acts on UPR pathways as well as others involved in DNA replication and the regulation of gene expression. It's binding to an RNA-binding protein called upstream-of-N-Ras protein (UNR) also activates Kruppel-like factor 13, a tumor suppressor and T cell differentiation regulator, and the role of this pathway in reversing inflammation remains to be established.
A feature of concern is the severe weight loss with this drug, though it is much less in humans. This is an area being worked on at present.
The antiviral and anti-inflammatory properties of clofoctol, associated with its safety profile and unique pharmacokinetics make a strong case for proposing clofoctol as an affordable therapeutic candidate for the treatment of COVID-19 patients… in resource poor settings."