Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors.

Through antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellent in vitro and in vivo properties have been identified. To evaluate the in vivo efficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients.

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

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.

Novel 2-Substituted 7-Azaindole and 7-Azaindazole Analogues as Potential Antiviral Agents for the Treatment of Influenza.

JNJ-63623872 (2) is a first-in-class, orally bioavailable compound that offers significant potential for the treatment of pandemic and seasonal influenza. Early lead optimization efforts in our 7-azaindole series focused on 1,3-diaminocyclohexyl amide and urea substitutions on the pyrimidine-7-azaindole motif. In this work, we explored two strategies to eliminate observed aldehyde oxidase (AO)-mediated metabolism at the 2-position of these 7-azaindole analogues. Substitution at the 2-position of the azaindole ring generated somewhat less potent analogues, but reduced AO-mediated metabolism. Incorporation of a ring nitrogen generated 7-azaindazole analogues that were equipotent to the parent 2-H-7-azaindole, but surprisingly, did not appear to improve AO-mediated metabolism. Overall, we identified multiple 2-substituted 7-azaindole analogues with enhanced AO stability and we present data for one such compound (12) that demonstrate a favorable oral pharmacokinetic profile in rodents. These analogues have the potential to be further developed as anti-influenza agents for the treatment of influenza.

Discovery of Novel, Orally Bioavailable ß-Amino Acid Azaindole Inhibitors of Influenza PB2.

In our efforts to develop novel small-molecule inhibitors for the treatment of influenza, we utilized molecular modeling and the X-ray crystal structure of the PB2 subunit of the influenza polymerase to optimize a series of acyclic ß-amino acid inhibitors, highlighted by compound 4. Compound 4 showed good oral exposure in both rat and mouse. More importantly, it showed strong potency versus multiple influenza-A strains, including pandemic 2009 H1N1 and avian H5N1 strains and showed a strong efficacy profile in a mouse influenza model even when treatment was initiated 48 h after infection. Compound 4 offers good oral bioavailability with great potential for the treatment of both pandemic and seasonal influenza.

Discovery of a novel, first-in-class, orally bioavailable azaindole inhibitor (VX-787) of influenza PB2.

In our effort to develop agents for the treatment of influenza, a phenotypic screening approach utilizing a cell protection assay identified a series of azaindole based inhibitors of the cap-snatching function of the PB2 subunit of the influenza A viral polymerase complex. Using a bDNA viral replication assay (Wagaman, P. C., Leong, M. A., and Simmen, K. A. Development of a novel influenza A antiviral assay. J. Virol. Methods 2002, 105, 105-114) in cells as a direct measure of antiviral activity, we discovered a set of cyclohexyl carboxylic acid analogues, highlighted by VX-787 (2). Compound 2 shows strong potency versus multiple influenza A strains, including pandemic 2009 H1N1 and avian H5N1 flu strains, and shows an efficacy profile in a mouse influenza model even when treatment was administered 48 h after infection. Compound 2 represents a first-in-class, orally bioavailable, novel compound that offers potential for the treatment of both pandemic and seasonal influenza and has a distinct advantage over the current standard of care treatments including potency, efficacy, and extended treatment window.

Natural prevalence of hepatitis C virus variants with decreased sensitivity to NS3.4A protease inhibitors in treatment-naive subjects.

BACKGROUND: The prevalence and clinical implications of naturally occurring variants that are resistant to hepatitis C virus (HCV) protease inhibitors in treatment-naive patients has not been reported. We report here the prevalence of such variants and their effect on clinical response. METHODS: Population sequence analysis of the NS3.4A protease was conducted in 570 treatment-naive subjects. RESULTS: Most subjects (98%) had wild-type virus. The remaining subjects had the following variants present in significant proportions (100%): V36M, 0.9%; R155K, 0.7%; V170A, 0.2%; and R109K, 0.2%. The V36M, R109K, and V170A substitutions confer low-level resistance (<7-fold) to protease inhibitors in replicon cells. The R155K substitution confers low-level resistance to telaprevir (TVR) and boceprevir and confers high-level resistance (>70-fold) to BILN 2061 and ITMN-191. Five subjects with the V36M or R109K variant were treated with 8-24 weeks of TVR and peginterferon-alpha2a (P) with or without ribavirin (R). Four achieved a sustained viral response, and 1 was lost to follow-up. In subjects with the R155K variant, TVR/PR provided greater antiviral activity than PR alone; however, the antiviral response was lower than that observed in subjects with wild-type virus. CONCLUSION: High levels of naturally occurring protease inhibitor-resistant variants were uncommon (<1% each) in HCV treatment-naive patients. TVR/PR efficiently inhibited V36M and R109K variants and contributed partial antiviral activity against the R155K variant. As new HCV agents are evaluated in clinical trials, it will be important to monitor the effect of baseline variants on sensitivity.

Preclinical profile of VX-950, a potent, selective, and orally bioavailable inhibitor of hepatitis C virus NS3-4A serine protease.

VX-950 is a potent, selective, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3-4A serine protease, and it demonstrated excellent antiviral activity both in genotype 1b HCV replicon cells (50% inhibitory concentration [IC50] = 354 nM) and in human fetal hepatocytes infected with genotype 1a HCV-positive patient sera (IC50 = 280 nM). VX-950 forms a covalent but reversible complex with the genotype 1a HCV NS3-4A protease in a slow-on, slow-off process with a steady-state inhibition constant (K(i)*) of 7 nM. Dissociation of the covalent enzyme-inhibitor complex of VX-950 and genotype 1a HCV protease has a half-life of almost an hour. A >4-log10 reduction in the HCV RNA levels was observed after a 2-week incubation of replicon cells with VX-950, with no rebound of viral RNA observed after withdrawal of the inhibitor. In several animal species, VX-950 exhibits a favorable pharmacokinetic profile with high exposure in the liver. In a recently developed HCV protease mouse model, VX-950 showed excellent inhibition of HCV NS3-4A protease activity in the liver. Therefore, the overall preclinical profile of VX-950 supports its candidacy as a novel oral therapy against hepatitis C.