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

Abstract

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 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 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.

Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase

“…A total of 141,936 compounds were screened for inhibitory activity against recHPIV3-JS NanoLuc (MOI = 0.2 TCID 50 units/cell). HTS was carried out analogous to our previous reports 64 , 73 , with automated reading of plates 30 hours after infection. Raw data were imported into the MScreen IT environment and analyzed using data mining tools developed in-house or build into the MScreen package as described 64 , 73 .…”

“…HTS was carried out analogous to our previous reports 64 , 73 , with automated reading of plates 30 hours after infection. Raw data were imported into the MScreen IT environment and analyzed using data mining tools developed in-house or build into the MScreen package as described 64 , 73 . Hit candidates were defined as compounds with anti-HPIV3 active >2.66×SD in control-dependent mean percent inhibition calculation and >2.1×SD in control-independent mean robust z-score calculation.…”

Structural Insight into Paramyxovirus and Pneumovirus Entry Inhibition

“…The existence of these pan-resistance sites suggests that it may be challenging to proactively counteract viral escape through synthetic scaffold optimization [69]. Supporting this notion, a large-scale high-throughput drug screen using a recombinant RSV strain carrying a signature pan-resistance mutation did not return any hits blocking F protein activity, although entry inhibitors typically emerge readily and are often pharmacodominant in anti-RSV drug screens [70,71]. Secondly, studies in the mouse RSV pathogenesis model have demonstrated early that signature pan-resistance mutations such as F D401E do not mandatorily coincide with viral attenuation in vivo [68], raising substantial concern that entry inhibitor-resistant RSV may emerge rapidly in the field, and remain pathogenic and possibly able to spread.…”

“…Alternatively, allosteric and substrate-analog inhibitors of the RSV polymerase complex have been identified [70,[101][102][103][104][105], some of which likewise showed promising drug-like properties and potently blocked virus replication in cell culture and in vivo. Ultimately, the therapeutic targeting of the polymerase may be more fruitful for two main reasons [106]: (i) all enzymatic functions of the viral polymerase complex, initiation of RNA synthesis, RNA elongation, mRNA capping, and cap methylation, must be performed multiple times to complete an individual viral replication cycle, providing-in contrast to entry inhibition-repeated opportunities for interference, and (ii) polymerase blockers suppress not only the synthesis of structural virion components directly, but also heighten virus susceptibility to the innate host antiviral response, since they prevent the expression of viral non-structural immune-modulatory proteins that are essential for viral pathogenesis [107,108].…”

Immune-Modulation by the Human Respiratory Syncytial Virus: Focus on Dendritic Cells

“…hRSV is an enveloped, negative-sense, and single-stranded RNA virus that encodes 10 genes that are translated into 11 proteins (Figure 1) (23). Its replication and gene transcription occur in the cytoplasm, thanks to the aid of an RNA-dependent RNA-polymerase that is encoded within the viral genome by the L gene (24, 25). For its adequate function, the L protein requires the viral phosphoprotein P, which associates to this RNA polymerase (26, 27).…”