Orally Efficacious Broad-Spectrum Ribonucleoside Analog Inhibitor of Influenza and Respiratory Syncytial Viruses

Abstract

Morbidity and mortality resulting from influenza-like disease are a threat, especially for older adults. To improve case management, next-generation broad-spectrum antiviral therapeutics that are efficacious against major drivers of influenza-like disease, including influenza viruses and respiratory syncytial virus (RSV), are urgently needed. Using a dual-pathogen high-throughput screening protocol for influenza A virus (IAV) and RSV inhibitors, we have identified N⁴-hydroxycytidine (NHC) as a potent inhibitor of RSV, influenza B viruses, and IAVs of human, avian, and swine origins. Biochemical in vitro polymerase assays and viral RNA sequencing revealed that the ribonucleotide analog is incorporated into nascent viral RNAs in place of cytidine, increasing the frequency of viral mutagenesis. Viral passaging in cell culture in the presence of an inhibitor did not induce robust resistance. Pharmacokinetic profiling demonstrated dose-dependent oral bioavailability of 36 to 56%, sustained levels of the active 5'-triphosphate anabolite in primary human airway cells and mouse lung tissue, and good tolerability after extended dosing at 800 mg/kg of body weight/day. The compound was orally efficacious against RSV and both seasonal and highly pathogenic avian IAVs in mouse models, reducing lung virus loads and alleviating disease biomarkers. Oral dosing reduced IAV burdens in a guinea pig transmission model and suppressed virus spread to uninfected contact animals through direct transmission. Based on its broad-spectrum efficacy and pharmacokinetic properties, NHC is a promising candidate for future clinical development as a treatment option for influenza-like diseases.

Keywords: antiviral agents; influenza; nucleoside analogs; respiratory syncytial virus.

FIG 1

The ribonucleoside analog NHC blocks negative-strand RNA viruses associated with influenza-like diseases. (A) Simultaneous anti-RSV and anti-IAV screen of a ribonucleoside analog library, carried out in triplicate. Shown are individual biological replicates (gray symbols) and mean values (black lines) ± SD. Hit candidates are highlighted in color, with a hit cutoff of ≥80% inhibition. (B) Structure of the NHC hit candidate. (C to G) For all dose-response measurements, symbols represent means ± SD of data from at least three biological repeats, expressed relative to values for vehicle (DMSO)-treated controls. EC₅₀ and CC₅₀ values were calculated through four-parameter variable-slope regression modeling, with 95% confidence intervals in brackets. (C) NHC activity against two RSV isolates on HEp-2 cells. Virus titration was performed by a plaque assay. PrestoBlue reagent was used to determine the effect of treatment on cell metabolic activity. (D) NHC activity against viruses representing group 1 and 2 HAs on MDCK cells. Virus titration was performed by HA assay and is expressed as relative HA units per milliliter (HAU/ml). (E) Efficacy of NHC against an HPAIV and an emerging AIV subtype on MDCK cells. Virus titration was performed by a plaque assay. (F) Efficacy of NHC against IBVs representing both currently circulating lineages on MDCK cells. Virus titration was performed by TCID₅₀ assays. (G) Comparison of NHC and T-705 antiviral efficacies on primary human bronchial tracheal epithelial cells (HBTEC) versus immortalized (im.) MDCK or HEp-2 cells. Virus replication assessment was based on virus-encoded luciferase reporter activity. Analysis was performed by two-way ANOVA, and P values are shown (ns, not significant).

FIG 2

Mechanistic assessment of NHC. (A) Time-of-addition variation assays. (Left) MDCK cells were infected with IAV-WSN-nanoLuc and treated with 10 μM NHC, 50 μM T-705 (positive-control influenza virus RdRp inhibitor) (57), or 10 μM GRP-71271 (positive-control IAV-WSN entry inhibitor) (32) at different times relative to infection. (Right) Hep2 cells were infected with recRSV-A2-L19F-fireSMASh (recombinant RSV) and treated with 10 μM NHC, 10 μM ALS-8176 (positive-control RSV RdRp inhibitor) (29), or 10 μM CL-309623 (positive-control RSV entry inhibitor) (82) at different times relative to infection. Reporter activity is expressed relative to values for vehicle-treated control infections (dashed lines; volume-equivalent DMSO was added to the controls two hours before infection [t₋₂]). Values represent means of data from three biological repeats ± SD. (B) NHC activity in dose-response minigenome assays. 293T cells were transiently transfected with IAV-WSN (H1N1), A/Vietnam (H5N1), A/Anhui (H7N9), or RSV minigenome systems. Increasing concentrations of NHC were added to the cells immediately after transfection. Luciferase reporter activity was measured after a 30-h exposure and is expressed relative to values for vehicle-treated wells. Means of data from three biological repeats ± SD are shown. Analysis was done by four-parameter variable-slope regression modeling. (C) Effect of NHC-TP on human DNA polymerase α activity. In vitro polymerase assays were carried out in the presence of a range of NHC-TP concentrations or aphidicolin for reference. Symbols represent mean values ± SD of data from 3 biological repeats each. (D) In cellula nucleoside competition assay. MDCK cells infected with WSN-nanoLuc (left) or RSV-fireSMASh (right) were exposed to 10 μM NHC and increasing concentrations of exogenously added natural nucleosides at the onset of infection. Values were normalized to the values for vehicle-treated controls and show means of data from three biological repeats ± SD. (E) In vitro RSV RdRp activity assay. Purified RSV P-L complexes were incubated with a 25-mer RNA oligonucleotide template and rNTPs (ribonucleotide triphosphate; lacking CTP) with an [α-³²P]UTP tracer. NHC (blue), NHC-TP (red), or CTP (black) was added at the specified final concentrations. Controls lacked CTP or contained the inactive L_D811A mutant. Lengths of the reaction products were determined by using Tr 1-25 and Tr 3-25 (RNA size markers) standards, and the sequence of the predominant 23-mer amplicon originating from major initiation at the +3 position is shown on the right. (F) Transition mutation frequency in viral RNA. Total RNA was extracted from cells infected with WSN (top) or RSV (bottom) in the presence of 10 μM NHC or the vehicle. Treatment was started immediately at the time of infection. PB1- or L-encoding cDNA was subcloned, and at least 10 independent clones each were subjected to Sanger sequencing; at least 7,737 nucleotides were determined per virus and exposure condition. Statistical analysis was performed with Fisher's exact test.

FIG 3

Anabolism and PK/PD profiling of NHC. (A) Anabolism of NHC in primary HBTECs. Cells were incubated with 20 μM NHC for the indicated exposure times, and intracellular concentrations of NHC and anabolites (NHC–5′-monophosphate [NHC-MP] and NHC-TP) were determined by LC-MS/MS. (B) NHC anabolite stability in HBTECs. Cells grown in the presence of 20 μM NHC for 24 h were switched to drug-free medium, and anabolite concentrations were monitored over a 6-h period by LC-MS/MS. (C and D) Time-concentration profiles for plasma levels of NHC after a single i.p. (C) or oral (D) dose in mice (3 animals per time point) at the specified levels. Shown are PK/PD profiles after a single oral dose in mice (3 animals per time point) at the specified levels. (E and F) Lung tissue concentrations of NHC (E) and of the bioactive NHC-TP anabolite (F) after a single oral NHC dose at the specified levels. Samples in panels A to F were analyzed by LC-MS/MS, and symbols represent mean values ± SD of data from 3 biological repeats each.

FIG 4

In vivo efficacy of NHC. (A to F) BALB/cJ mice were infected i.n. with 1 × 10⁵ PFU of RSV-A2-L19F (A and B) or 1,000 PFU of IAV-PR8 (C to F), and NHC was dosed orally b.i.d. Symbols represent individual biological repeats, and columns show mean values ± SD. LOD, limit of detection. (A) Lung RSV loads were determined at 5 days postinfection through immunoplaque assays. Dosing was initiated prophylactically at 2 h preinfection. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test. (B) Breath distension of peripheral arteries was quantified 8 days after infection with RSV through pulse oximetry. Control animals were mock infected and vehicle treated. Statistical significance was determined by one-way ANOVA with Tukey's post hoc test. (C) Lung IAV-PR8 loads were determined at 6 days postinfection. Dosing was initiated prophylactically. Statistical significance was determined by one-way ANOVA with Dunnett's post hoc test. (D) Comparison of NHC with the SOC oseltamivir. Lung IAV-PR8 loads were determined on the specified days postinfection. Dosing was initiated prophylactically. Statistical significance was determined by two-way ANOVA with Tukey's post hoc test. (E) Relative expression levels of the proinflammatory cytokines IFN-γ and IL-6 in lung tissue of IAV-PR8-infected animals were quantified by RT-qPCR at 3 days postinfection and are expressed relative to values for uninfected, vehicle-treated animals. Statistical significance was determined by one-way ANOVA with Dunnett's post hoc test. (F) Postexposure dosing of NHC, initiated at 6 h postinfection. Lung IAV-PR8 loads were determined at 3 and 6 days postinfection. Statistical significance was determined by unpaired t tests with Welch's correction for each time point. (G) Oral efficacy of NHC against HPAIV. Mice were infected with 6 PFU of A/Vietnam/1203/2004 (H5N1), and brain and lung virus loads were determined at 6 days postinfection. Treatment was initiated prophylactically at 2 h preinfection; statistical significance was determined by one-way ANOVA with Tukey's post hoc test. (H and I) Oral NHC efficacy and suppression of virus transmission in guinea pigs. (H) Source animals were infected i.n. with 10⁴ PFU of IAV-NL/09, and virus loads in nasal lavage fluids were determined on days 1, 3, 5, and 7 postinfection. Treatment was initiated prophylactically at 2 h preinfection and continued b.i.d. to the end of day 3 postinfection. (I) Untreated and uninfected contact animals were added at 24 h postinfection, and transmitted virus titers in nasal lavage fluids were determined on days 2, 4, 6, and 8. Analysis was done with two-way ANOVA and Dunnett's post hoc test.