New research published in Science reports 4’-Fluorouridine (4′-FlU) as an oral antiviral against the respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, the researchers described its inhibition against viruses in cell lines and organoids and demonstrated the high efficacy of 4’GIU in daily oral treatments in animal models.
Using in vitro ribonucleic acid (RNA)-dependent RNA polymerase (RdRP) assays, the researchers established that the 4′-FIU stalled transcription through polymerase inhibition. Based on these results, the researchers defined 4′-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.
Study: 4′-Fluorouridine is an oral antiviral that blocks respiratory syncytial virus and SARS-CoV-2 replication. Image Credit: fizkes / Shutterstock.com
Introduction
The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2, has underscored the importance of having an orally bioavailable broad-spectrum antiviral that is also used pre-emptively rather than a delayed treatment option.
The development of broad-spectrum antivirals is essential to have a significant effect on a growing pandemic, specifically before a new pathogen emerges. In this context, the researchers identified RSV disease to test 4’FIU against as a result of the major health threat imposed by RSV and the lack of clinical trials for anti-RSV therapeutics. RSV disease primarily affects children below 5 years and the elderly population, with no licensed therapeutics currently for its treatment.
Molnupiravir is an approved drug against COVID-19. Exploring the 4′-FIU substitutions in a series of analogs of the molnupiravir parent molecule N4-hydroxycytidine (NHC), the researchers arrived at 4’-FIU, which is a ribonucleoside analog.
The researchers chose 4’FIU because it has been reported to act against RSV, SARS-CoV-2, and related viruses. Furthermore, 4’FIU is associated with several advantages including the small atomic radius and strong stereo-electronic effect of fluorine, increased lipophilicity, and greater metabolic stability.
Study findings
The researchers report that 4′-FlU is a potent broad-spectrum antiviral with high selectivity index. Testing a representative of phylogenetically distant positive-sense RNA viruses, the researchers found that the betacoronavirus SARS-CoV-2 was also sensitive to 4′-FlU. The EC50 values ranged from 0.2 to 0.6 micromolar (μM) against isolates of different lineages.
Probing the mechanism of activity using polymerase inhibition within in vitro RdRP assays, the researchers found that 4′-FlU induces a sequence-modulated delayed stalling of RSV and SARS-CoV-2 RdRP. Because 4′-FlU also triggers immediate RdRP stalling, depending on the sequence, it suggests steric hindrance of polymerase advance or of accommodating the next incoming nucleotide as the underlying mechanism of activity.
Notably, SARS-CoV-2 is observed to be lower in sensitivity to 4′-FlU as compared to RSV. This could be because of the exonuclease activity of the coronavirus polymerase against 4′-FlU.
The researchers reported that the 4′-FlU is rapidly 'anabolized' and is metabolically stable in the human airway epithelium (HAE) organoids/cultures. They cultured the HAEs such that it was a well-differentiated three-dimensional (3D) airway epithelium that included ciliated and mucous-producing cells. Taken together, the 4’-FIU was potently antiviral.
To test in vivo, the researchers used mouse models of RSV infection and found that the 4′-FlU is orally efficacious in a therapeutic dosing regimen. Based on these observations, the researchers reported that the therapeutic window of 4′-FlU extended to 24 hours after infection in mice.
Up to 24 (RSV) or 12 (SARS-CoV-2) hours after infection with daily oral administration of 4’-FIU in mice and ferrets, the researchers observed a significant reduction in the viral burden. Because the invasion of RSV and SARS-CoV-2 in hosts such as humans is slower than these live models, the researchers believe that the findings from this study positively outline a viable therapeutic window for human treatment with 4’-FIU.
Further, they found that 4′-FlU is efficacious against SARS-CoV-2 replication in HAE organoids. The researchers also tested against an early pandemic isolate (WA1) and several SARS-CoV-2 variants of concern (VOCs) including the Alpha, Gamma, and Delta variants in the ferret model. All viruses were found to be highly sensitive to 4’-FIU.
The researchers observed that the viral titers in nasal turbinate tissue and associated viral RNA copy numbers correlated with the reduction in shed virus load. Notably, there was no observed shedding of infectious particles in all animals after 2.5 days of treatment, or 3 days post-infection.
While the tolerability studies are under study, the researchers observed that the 4′-FlU was well tolerated by the human organoid models and was efficacious in the animal models.
Conclusions
The current study has identified and characterized 4′-FlU as a potent inhibitor of different clinically relevant RNA virus families. The researchers showed that 4’FIU causes delayed stalling of RSV and SARS-CoV-2 polymerases in the in vitro RdRP assays, similar to remdesivir.
The current study also demonstrated potent activity against the SARS-CoV-2 Alpha, Delta and Gamma, VOCs, thereby demonstrating the broad anti-coronavirus efficacy of 4′-FlU. These results reassure an effective activity of the compound against future SARS-CoV-2 VOCs that may be increasingly less responsive to vaccines or other therapeutics.
The current study establishes 4′-FlU as a broad-spectrum orally efficacious inhibitor of major RNA viruses. This drug may therefore become a promising therapeutic option for RSV disease and COVID-19.
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