Preclinical Characterization of PC786, an Inhaled Small-Molecule Respiratory Syncytial Virus L Protein Polymerase Inhibitor.

Although respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants and young children, attempts to develop an effective therapy have so far proved unsuccessful. Here we report the preclinical profiles of PC786, a potent nonnucleoside RSV L protein polymerase inhibitor, designed for inhalation treatment of RSV infection. PC786 demonstrated a potent and selective antiviral activity against laboratory-adapted or clinical isolates of RSV-A (50% inhibitory concentration [IC50], <0.09 to 0.71 nM) and RSV-B (IC50, 1.3 to 50.6 nM), which were determined by inhibition of cytopathic effects in HEp-2 cells without causing detectable cytotoxicity. The underlying inhibition of virus replication was confirmed by PCR analysis. The effects of PC786 were largely unaffected by the multiplicity of infection (MOI) and were retained in the face of established RSV replication in a time-of-addition study. Persistent anti-RSV effects of PC786 were also demonstrated in human bronchial epithelial cells. In vivo intranasal once daily dosing with PC786 was able to reduce the virus load to undetectable levels in lung homogenates from RSV-infected mice and cotton rats. Treatment with escalating concentrations identified a dominant mutation in the L protein (Y1631H) in vitro In addition, PC786 potently inhibited RSV RNA-dependent RNA polymerase (RdRp) activity in a cell-free enzyme assay and minigenome assay in HEp-2 cells (IC50, 2.1 and 0.5 nM, respectively). Thus, PC786 was shown to be a potent anti-RSV agent via inhibition of RdRp activity, making topical treatment with this compound a novel potential therapy for the treatment of human RSV infections.

Coates M ，Brookes D ，Kim YI ，Allen H ，Fordyce EAF ，Meals EA ，Colley T ，Ciana CL ，Parra GF ，Sherbukhin V ，Stockwell JA ，Thomas JC ，Hunt SF ，Anderson-Dring L ，Onions ST ，Cass L ，Murray PJ ，Ito K ，Strong P ，DeVincenzo JP ，Rapeport G ... -  《-》

Late therapeutic intervention with a respiratory syncytial virus L-protein polymerase inhibitor, PC786, on respiratory syncytial virus infection in human airway epithelium.

Effective anti-respiratory syncytial virus (RSV) agents are still not available for clinical use. Current major targets are virus surface proteins, such as a fusion protein involved in viral entry, but agents effective after RSV infection is established are required. Here we have investigated the effects of late therapeutic intervention with a novel inhaled RSV polymerase inhibitor, PC786, on RSV infection in human airway epithelium. Air liquid interface-cultured bronchial or small airway epithelium was infected with RSVA2. PC786 was applied apically or basolaterally once daily following peak virus load on Day 3 post inoculation. Apical wash was collected daily for determination of viral burden by PCR and plaque assay (primary endpoints) and biomarker analyses. The effects were compared with those of ALS-8112, an anti-RSV nucleoside analogue, and GS-5806, a fusion-protein inhibitor, which were treated basolaterally. Late intervention with GS-5806 did not show significant anti-viral effects, but PC786 produced potent, concentration-dependent inhibition of viral replication with viral load falling below detectable limits 3 days after treatment commenced in airway epithelium. These effects were superior to those of ALS-8112. PC786 showed inhibitory activities against RSV-induced increases of CCL5, IL-6, double-strand DNA and mucin. The effects of PC786 were also confirmed in small airway epithelium. Late therapeutic intervention with the RSV polymerase inhibitor, PC786, reduced the viral burden quickly in human airway epithelium. Thus, PC786 demonstrates the potential to be an effective therapeutic agent to treat active RSV infection.

Brookes DW ，Coates M ，Allen H ，Daly L ，Constant S ，Huang S ，Hows M ，Davis A ，Cass L ，Ayrton J ，Knowles I ，Strong P ，Rapeport G ，Ito K ... -  《-》

Differential antiviral activities of respiratory syncytial virus (RSV) inhibitors in human airway epithelium.

Mirabelli C ，Jaspers M ，Boon M ，Jorissen M ，Koukni M ，Bardiot D ，Chaltin P ，Marchand A ，Neyts J ，Jochmans D ... -  《-》

Comparative Therapeutic Potential of ALX-0171 and Palivizumab against Respiratory Syncytial Virus Clinical Isolate Infection of Well-Differentiated Primary Pediatric Bronchial Epithelial Cell Cultures.

Respiratory syncytial virus (RSV) causes severe lower respiratory tract infections in young infants. There are no RSV-specific treatments available. Ablynx has been developing an anti-RSV F-specific nanobody, ALX-0171. To characterize the therapeutic potential of ALX-0171, we exploited our well-differentiated primary pediatric bronchial epithelial cell (WD-PBEC)/RSV infection model, which replicates several hallmarks of RSV disease in vivo Using 2 clinical isolates (BT2a and Memphis 37), we compared the therapeutic potential of ALX-0171 with that of palivizumab, which is currently prescribed for RSV prophylaxis in high-risk infants. ALX-0171 treatment (900 nM) at 24 h postinfection reduced apically released RSV titers to near or below the limit of detection within 24 h for both strains. Progressively lower doses resulted in concomitantly diminished RSV neutralization. ALX-0171 was approximately 3-fold more potent in this therapeutic RSV/WD-PBEC model than palivizumab (mean 50% inhibitory concentration [IC50] = 346.9 to 363.6 nM and 1,048 to 1,090 nM for ALX-0171 and palivizumab, respectively), irrespective of the clinical isolate. The number of viral genomic copies (GC) was determined by quantitative reverse transcription-PCR (RT-qPCR), and the therapeutic effect of ALX-0171 treatment at 300 and 900 nM was found to be considerably lower and the number of GCs reduced only moderately (0.62 to 1.28 log10 copies/ml). Similar findings were evident for palivizumab. Therefore, ALX-0171 was very potent at neutralizing RSV released from apical surfaces but had only a limited impact on virus replication. The data indicate a clear disparity between viable virus neutralization and GC viral load, the latter of which does not discriminate between viable and neutralized RSV. This report validates the RSV/WD-PBEC model for the preclinical evaluation of RSV antivirals.

Broadbent L ，Parke HG ，Ferguson LJ ，Millar A ，Shields MD ，Detalle L ，Power UF ... -  《-》

Development of an allosteric inhibitor class blocking RNA elongation by the respiratory syncytial virus polymerase complex.

Respiratory syncytial virus (RSV) represents a significant health threat to infants and to elderly or immunocompromised individuals. There are currently no vaccines available to prevent RSV infections, and disease management is largely limited to supportive care, making the identification and development of effective antiviral therapeutics against RSV a priority. To identify effective chemical scaffolds for managing RSV disease, we conducted a high-throughput anti-RSV screen of a 57,000-compound library. We identified a hit compound that specifically blocked activity of the RSV RNA-dependent RNA polymerase (RdRp) complex, initially with moderate low-micromolar potency. Mechanistic characterization in an in vitro RSV RdRp assay indicated that representatives of this compound class block elongation of RSV RNA products after initial extension by up to three nucleotides. Synthetic hit-to-lead exploration yielded an informative 3D quantitative structure-activity relationship (3D-QSAR) model and resulted in analogs with more than 20-fold improved potency and selectivity indices (SIs) of >1,000. However, first-generation leads exhibited limited water solubility and poor metabolic stability. A second optimization strategy informed by the 3D-QSAR model combined with in silico pharmacokinetics (PK) predictions yielded an advanced lead, AVG-233, that demonstrated nanomolar activity against both laboratory-adapted RSV strains and clinical RSV isolates. This anti-RSV activity extended to infection of established cell lines and primary human airway cells. PK profiling in mice revealed 34% oral bioavailability of AVG-233 and sustained high drug levels in the circulation after a single oral dose of 20 mg/kg. This promising first-in-class lead warrants further development as an anti-RSV drug.

Cox RM ，Toots M ，Yoon JJ ，Sourimant J ，Ludeke B ，Fearns R ，Bourque E ，Patti J ，Lee E ，Vernachio J ，Plemper RK ... -  《-》