Preclinical activity of VX-787, a first-in-class, orally bioavailable inhibitor of the influenza virus polymerase PB2 subunit

Abstract

VX-787 is a novel inhibitor of influenza virus replication that blocks the PB2 cap-snatching activity of the influenza viral polymerase complex. Viral genetics and X-ray crystallography studies provide support for the idea that VX-787 occupies the 7-methyl GTP (m(7)GTP) cap-binding site of PB2. VX-787 binds the cap-binding domain of the PB2 subunit with a KD (dissociation constant) of 24 nM as determined by isothermal titration calorimetry (ITC). The cell-based EC50 (the concentration of compound that ensures 50% cell viability of an uninfected control) for VX-787 is 1.6 nM in a cytopathic effect (CPE) assay, with a similar EC50 in a viral RNA replication assay. VX-787 is active against a diverse panel of influenza A virus strains, including H1N1pdm09 and H5N1 strains, as well as strains with reduced susceptibility to neuraminidase inhibitors (NAIs). VX-787 was highly efficacious in both prophylaxis and treatment models of mouse influenza and was superior to the neuraminidase inhibitor, oseltamivir, including in delayed-start-to-treat experiments, with 100% survival at up to 96 h postinfection and partial survival in groups where the initiation of therapy was delayed up to 120 h postinfection. At different doses, VX-787 showed a 1-log to >5-log reduction in viral load (relative to vehicle controls) in mouse lungs. Overall, these favorable findings validate the PB2 subunit of the viral polymerase as a drug target for influenza therapy and support the continued development of VX-787 as a novel antiviral agent for the treatment of influenza infection.

FIG 1

Chemical structure of m⁷GTP and VX-787.

FIG 2

Binding data for VX-787 to PB2 cap-binding domain. (A) Isothermal titration calorimetric data: the upper panel shows raw ITC data, and the lower panel shows integrated ITC data fitted to a single-binding-site model. (B) Surface plasmon resonance (SPR) binding data and kinetic/steady-state analysis for VX-787 with the PB2 cap-binding domain.

FIG 3

X-ray structure of PB2 cap-binding domain. (A) Contact residues with m⁷GTP (left) or VX-787 (right). (B) Ribbon diagram and surface depicting cap-binding domain of PB2 bound to VX-787 (PyMOL Molecular Graphics System, version 1.7; Schrödinger, LLC).

FIG 4

Time-of-addition experiment. MDCK cells were infected with influenza A/Puerto Rico/8/34 virus at an MOI of 2. Arrows indicate times at which test compounds were added, at 45 min (left) or 6 h (right) postinfection. The concentrations and compounds tested were 0.04 μM VX-787, 50 μM favipiravir, 22 μM oseltamivir carboxylate, and 1 μM zanamivir, each approximately 25-fold the EC₅₀ in a 3-day CPE assay. Additionally, the concentrations of oseltamivir carboxylate and zanamivir chosen were >1,000-fold the enzymatic IC₅₀ for these compounds. Samples were harvested every 2 or 3 h posttreatment to monitor the levels of viral (+)-strand RNA and viral (−)-strand RNA using a bDNA assay.

FIG 5

Evaluation of VX-787 cytopathic effect. MDCK cells were infected with influenza A/Puerto Rico/8/34 virus at an MOI of 2 in the presence of inhibitor. The infected cells were harvested at different times postinfection to determine cell viability by analysis of ATP content using the CPE assay (A) or stained with Coomassie blue in a plaque assay (B). Viability is graphed as percent means ± standard deviations of the results of three independent experiments.

FIG 6

Antiviral activity of VX-787 (left panel) and oseltamivir carboxylate (right panel) against influenza A/Puerto Rico/8/34 virus in MDCK cells was determined across a range of MOIs. MDCK cells in 384-well plates were infected with the indicated range of virus and incubated with 3-fold dilutions of VX-787 ranging from 0.00015 to 1 μM or with oseltamivir carboxylate at from 0.0076 to 50 μM. After incubation at 37°C in 5% CO₂ for 3 days, the antiviral activity was investigated by analysis of ATP levels of infected cells. Results are presented as the mean percentage values normalized to noninfected controls, obtained from quadruplicate wells in the same experiment.

FIG 7

In vitro antiviral combination studies with VX-787 and oseltamivir carboxylate or zanamivir. Inhibitor pairs were tested in full factorial combination, and the expected additive antiviral effect of each combination was calculated according to the Bliss independence method. Data shown were assessed at the 95% confidence level. The simple additivity result is indicated by the zero plane, on the vertical axis. Synergy is indicated by values above the additivity plane and antagonism by values below the plane.

FIG 8

Prophylactic or delayed-start-to-treat effectiveness of VX-787 in the mouse influenza A virus infection model. Male BALB/c mice (8/group) were infected with a lethal challenge of influenza virus (A/Puerto Rico/8/34; 3 × 10⁵ TCID₅₀/mouse) followed by administration of vehicle, oseltamivir, or VX-787 at the indicated doses and start times. The 21-day survival rate and percent BW loss (means ± standard errors of the means [SEM]) are shown. The shaded area represents the treatment period. Oseltamivir was not included in studies beyond 48 h postinfection.

FIG 9

Prophylactic or delayed-start-to-treat effectiveness of VX-787 in the mouse influenza A/Viet Nam/1203/2004 (H5N1) virus infection model. Male BALB/c mice (8/group) were infected with a lethal challenge of influenza virus followed by administration of vehicle, oseltamivir, or VX-787 at the indicated doses and start times. The 21-day survival rate and percent BW loss (means ± SEM) are shown. The shaded area represents the treatment period.