4′-FlU: novel broad-spectrum antiviral drug class with activity against SARS-CoV-2


The COVID-19 pandemic and resurgence of infections by other respiratory RNA viruses such as respiratory syncytial virus (RSV) in children has caused an urgent need for the development of orally available broad-spectrum antiviral therapeutics.

In a study published online on Dec. 2 in Science, researchers in the Institute for Biomedical Sciences at Georgia State University report a new candidate ribonucleoside analog, 4′-fluorouridine (4′-FlU), that has potent antiviral activity against SARS-CoV-2, RSV and other respiratory RNA viruses in cell culture, human organoids and different animal models when administered orally once-daily.

“Mechanistically, we show that 4′-FlU is in a different class from molnupiravir that is currently considered for regulatory approval,” said Dr. Richard Plemper, senior author of the study, Distinguished University Professor and director of the Center for Translational Antiviral Research at Georgia State.

“4′-FlU does not act as a mutagen but induces termination of the viral polymerase, aborting replication of the viral genome. There is an urgent need to expand the therapeutic arsenal against SARS-CoV-2 and 4′-FlU has strong developmental promise as a companion drug.”

In the study, 4′-FlU was tested against different SARS-CoV-2 variants of concern in ferrets, which have emerged as a leading model for drug testing, and against respiratory syncytial virus in mice.

The researchers found that this drug potently blocked SARS-CoV-2 replication including the gamma and delta variants in the ferret, and efficiently suppressed RSV burden in mouse lungs.

“We are excited that 4′-FlU is the only orally available antiviral candidate currently developed against SARS-CoV-2 that is active when given once-daily,” said Dr. Julien Sourimant, first author of the study and a researcher in Dr. Plemper’s lab in the Institute for Biomedical Sciences, “which should be a major asset in ensuring outpatient compliance.”

This research on 4′-FlU emerged from a collaboration of the team at Georgia State University with researchers at Emory University and the Texas Biomedical Research Institute.

The study was funded by public health service grants from the National Institutes of Health/National Institute of Allergy and Infectious Diseases to Georgia State University.

Co-authors of the study include J. Sourimant, C.M. Lieber, M. Aggarwal, R.M. Cox, J.D. Wolf, J.-J. Yeong, M. Toots and R.K. Plemper at Georgia State University; C. Ye and L. Martinez-Sobrido at Texas Biomedical Research Institute; and Z. Sticher, A.A. Kolykhalov, G.R. Bluemling, M.G. Natchus and G.R. Painter at Emory University.

4’-FlU is a broad-spectrum mononegavirus inhibitor with high SI

The compound showed potent dose-dependent activity against all RSV strains tested, returning half-maximal efficacious concentrations (EC50 values) ranging from 0.61 to 1.2 μM (Fig. 1B) (Table S1). This cell culture potency was on par with anti-RSV activity of previously reported NHC (17), the free base of molnupiravir that is currently in clinical development (Fig. S1).

Global metabolic activity of established human and animal cell lines (HEp-2, MDCK, BHK-T7, BEAS-2B) exposed to up to 500 μM of 4’-FlU remained unaltered (Fig. 1C) (Table S2). When glucose was replaced with galactose as carbohydrate source to link cell metabolic activity strictly to mitochondrial oxidation (18), we determined a half-maximal cytotoxic concentration (CC50) of 4’-FlU of 250 μM (Fig. 1C) (Table S2).

When tested on disease-relevant primary human airway epithelial cells (HAE) derived from two different donors (Fig. 1D), 4’-FlU showed ≥17-fold increased anti-RSV potency but unchanged low cytotoxicity (CC50 169 μM) (Fig. 1E), resulting in a high selectivity index (SI = EC50/CC50) of ≥1877.

Consistent with these findings, in-cell quantitative immunocytochemistry confirmed that 4’-FlU similarly reduced steady-state levels of nuclear-(SDH-A; IC50 272.8 μM) and mitochondrial-(COX-I; IC50 146.8 μM) encoded proteins in HAEs only at high concentrations (Fig. S2).

Assessment of the broader 4’-FlU indication spectrum against a panel of related major pathogens of the mononegavirus order, including measles virus (MeV), human parainfluenza virus type 3 (HPIV3), Sendai virus (SeV), vesicular stomatitis virus (VSV), and rabies virus (RabV) consistently demonstrated sub-micromolar active concentrations (Fig. 1F) (Table S1).

The positive-sense RNA betacoronavirus SARS-CoV-2 was apparently only slightly less sensitive to 4’-FlU, with EC50 values ranging from 0.5 to 5.1 μM against isolates of different lineages (Fig. 1G) (Table S1).

An initial mechanistic characterization of 4’-FlU in cell-based minireplicon systems revealed inhibition of pneumovirus and paramyxovirus RdRP complex activity (Fig. 1H) (Table S1). In accordance with broad-spectrum activity against mononegavirus, the RdRP activity of Nipah virus (NiV), a highly pathogenic zoonotic paramyxovirus with strong pandemic potential (19), was also efficiently inhibited by 4’-FlU in an NiV minireplicon reporter assay.

The antiviral effect of 4’-FlU was dose-dependently reversed by addition of an excess of exogenous pyrimidines, cytidine and uridine, but not purines, to the cultured cells, which furthermore is consistent with competitive inhibition of RdRP activity (2) (Fig. 1I).

Fig. 1.
Fig. 1.4’-FlU is a potent antiviral with broad spectrum and high selectivity index.(A) Chemical structure of 4’-FlU. (B) Virus yield reduction of RSV clinical isolates 6A8, 16F10 (B antigenic subgroup), 2-20 and recombinant recRSV-A2line19F-[mKate] (A antigenic subgroup). (C) HEp-2, MDCK, BHK-T7 and BEAS-2B cells were assayed for reduction in cell metabolism after incubation with serial dilutions of 4’-FlU or vehicle. (D,E) Human airway epithelial (HAE) cells from “F1” and “M4” donors were incubated with serial dilutions of 4’-FlU or vehicle (DMSO) treatment (D), and either infected with recRSV-A2line19F-[FireSMASh] to assess viral inhibition through reporter activity or uninfected to determine cytotoxicity (E). (F) Dose-response curves of 4’-FlU against a panel of recombinant mononegaviruses. Viral inhibition determined by reporter activity. (G) Virus yield reduction of SARS-CoV-2 WA1/2020 and CA/2020 isolates after incubation with serial dilutions of 4’-FlU or vehicle. (H) Dose-response curves of 4’-FlU against transiently expressed viral polymerase complexes from mononegaviruses MeV, RSV, NiV or HPIV-3. (I) recRSV-A2line19F-[FireSMASh]-infected cells were treated with 10 μM of 4’-FlU and serial dilutions of exogenous nucleotides in extracellular media. Viral inhibition determined by reporter activity. Symbols represent independent repeats (B,E,G,H,I) or mean with standard deviation (C,F), and lines represent means.

reference link :https://www.biorxiv.org/content/10.1101/2021.05.19.444875v1.full

More information: Science (2021). DOI: 10.1126/science.abj5508


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