New favipiravir derivative is a potent SARS-CoV-2 inhibitor

By Michael Greenwood, M.Sc.May 27 2021

The search for potent drug inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been underway since the beginning of the coronavirus disease 2019 (COVID-19) pandemic. The antiviral influenza drug favipiravir has been indicated as a potential lead by researchers with some, so far, limited but promising clinical reports.

Favipiravir is an RNA-dependent RNA-polymerase (RdRp) enzyme inhibitor with a mechanism of action that depends on the structural similarity of the molecule with purine nucleotides. Thus, when mistakenly incorporated into the viral RNA sequence, it causes early termination. The drug causes lethal mutagenesis in influenza, though the broadly applicable mechanism of action allows the drug good activity against some other viruses.

The RdRp enzyme plays a critical role in SARS-CoV-2 replication and transcription while also being absent from human cells, meaning that the drug is likely to induce fewer off-target effects. Unfortunately, the drug has been shown to be detrimental to fetal development in pregnant women, and bears poor bioavailability properties, particularly in the lungs. Additionally, binding affinities between the SARS-CoV-2 form of the enzyme and favipiravir are poor, as determined by in silico measurements.

Study: Discovery of (E)-N-(4-cyanobenzylidene)- 6-fluoro- 3-hydroxypyrazine-2 -carboxamide (cyanorona-20): the first potent and specific anti-COVID-19 drug. Image Credit: joshimerbin / Shutterstock

In a new study recently published in Chemical Papers by Dr. Amgad Rabie (May 16^th, 2021), a high potency derivative of favipiravir towards SARS-CoV-2 is engineered and investigated.

Cyanorona-20

Following extensive molecular docking simulations with compound libraries of small molecules similar to favipiravir (E)-N-(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide was identified, termed cyanorona-20. This is a portmanteau between cyano, the major distinguishing functional group that the molecule bears, and coronavirus. The major aspect of the drug remains the potent antiviral favipiravir scaffold, with the modified components serving to improve biodistribution and bioavailability by balancing the lipophilic and hydrophilic characteristics of the molecule. This introduces greater structural stability and increases polarity and hydrogen bonding availability for better membrane penetration and interaction with proteins.

In silico modeling suggested that cyanorona-20 would interact in the same way as favipiravir with ribofuranosyl-5′-triphosphate  (RTP). This sugar moiety activates the drug inside the body and allows it to be incorporated into the RNA sequence. This complex is then able to go on to interact with the RdRp enzyme at the Asp760 active site, preventing further transcription by this enzyme. The affinity of cyanorona-20 towards this site is greater than that observed in favipiravir due to the cyano functional group, which induces severe instability in the RdRp residue by steric hindrance.

Cyanorona-20 was synthesized from favipiravir via condensation reaction with 4-cyanobenzaldehyde in equimolar concentrations in the presence of acetic acid, and as a tautomer favors the enol form in aqueous conditions. In vitro assays comparing the drug with favipiravir and four other candidates (GS-441524-TP, arbidol, remdesivir, and hydroxychloroquine) in Vero E6 cells demonstrated much lower effective concentrations were needed to successfully inhibit SARS-CoV-2 proliferation, with an EC₅₀ of only 0.45 μM compared with 94 and 20 μM for favipiravir and remdesivir, respectively. The concentration required for 100% effective viral inhibition was also the lowest of the five compounds tested, at 1.4 μM, while the concentration at which cell death occurred was well over 100 μM.

The author points out that the very highly tolerated drug should be safe for human use, while the minute concentrations required to suppress virus replication are highly promising. It is also stated that the improved mechanism of inhibition of the molecule over favipiravir may allow less chance of resistance developing in SARS-CoV-2, as the conformational features of viral proteins are essential to functional replication, and thus unlikely to alter significantly.

However, the full mechanism of action behind SARS-CoV-2 inhibition by cyanorona-20 is yet to be elucidated, with six possible routes being suggested that may act independently or simultaneously: misincorporation into RNA strands, preventing further elongation; evasion of proofreading viral proteins, lowering RNA production; competitive binding to conserved polymerase domains, blocking transcription; induction of chain termination in RdRp; or lethal mutagenesis, lowering virus fitness.