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 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.

Keywords: RNA-dependent RNA polymerase; RdRp; animal virus; antiviral agent; drug development; medicinal chemistry; respiratory disease; respiratory syncytial virus; viral polymerase.

Figure 1.

HTS Screening and hit identification. A, HTS campaign of 56,557 compounds conducted against recRSV-A2-L19F_D489E-fireSMASh. For each compound, robust z-scores and percentage inhibition values were calculated, and hit cutoff for each analysis method is shown (dashed lines). The inset summarizes counterscreening results obtained for 139 primary screen hit candidates that met both inclusion criteria. B, automated dose–response potency and cytotoxicity testing in 384-well format for 13 hit candidates advanced from A. Compounds with CC₅₀ <20 μm, SI <1, or EC₅₀ ≥5 μm were discontinued. Primary HTS and automated counterscreens in A and B were carried out in single biological repeats. C, structure of GRP-156784, the primary hit candidate passing all performance milestones. Numbers and dashed red circles denote scaffold sections modified individually for synthetic hit-to-lead development.

Figure 2.

Hit confirmation. A, inhibitory activity of resynthesized GRP-156784 against different luciferase reporter viruses (red y axis), standard RSV A2-L19F (blue y axis), and measurements of cellular metabolic activity of uninfected cells exposed to the compound (black y axis). Where applicable, active concentrations were calculated through four-parameter variable slope regression modeling; values in parentheses denote 95% confidence intervals). B, anti-RSV activity and cytotoxicity of resynthesized GRP-156784 in primary HBTECs. Symbols in A and B represent biological repeats (n = 3 each or greater); error bars, S.D.

Figure 3.

Mechanistic profiling of GRP-156784. A, time-of-addition variation studies against the RSV target. Compound GRP-156784 (red), RSV entry inhibitor BMS-433771 (blue), or viral polymerase inhibitor JMN3–003 (maroon) were added to equally infected cells at the indicated time points and concentrations. Relative virus replication in all cultures was determined 33 h after infection. B, dose–response minigenome reporter assay inhibition by GRP-156784. The compound was tested against RSV–, IAV-WSN–, and MeV–derived minigenomes. C, RT-qPCR quantitation of relative RSV N protein–encoding mRNA levels present in RSV-infected cells after incubation in the presence of GRP-156784, RSV ribonucleoside analog inhibitor ALIOS-8176, or vehicle volume equivalents. Statistical analysis through one-way analysis of variance with Dunnett's multiple-comparison post hoc test. Symbols in A–C represent individual biological repeats (n = 3 each); error bars, S.D. Previous characterized reference compounds BMS-433771 and JMN3–003 were analyzed in A in two biological repeats; error bars, range. In C, each of the three biological repeats was determined in technical duplicates.

Figure 4.

First-generation optimized leads and 3D-QSAR model for the hit scaffold. A, bioactivity of analogs AVG-094 and AVG-158 compared with resynthesized GRP-156784. No detectable cytotoxicity was measured for either AVG094 (blue dashed line) or AVG-158 (red dashed line). B, reduction of progeny RSV A2-L19F virus load by AVG-094 and AVG-158 determined in dose–response activity assays. Active concentrations were determined through four-parameter variable-slope regression modeling. LoD, level of detection. Symbols in A and B represent individual biological repeats (n = 3 each); error bars, S.D. C, in vitro RSV RdRp activity assay using synthetic template RNA and purified polymerase complexes conducted in the presence of AVG-094 or AVG-158. The compounds show dose-dependent block of 3′ RNA extension elongation after back-priming (back-priming extens. elong.) and de novo RNA synthesis from the promoter, but do not prevent extension by up to three nucleotides after back-priming (back-priming 3′ extens.). L_D811A is bio-inactive due to the alanine substitution in the L catalytic site and was included as assay specificity control. D, generation and testing of a 3D-QSAR model developed based on a subset of GRP-156784 analogs synthesized for chemical optimization of the scaffold. AutoGPA embedded in the MOE package was used for model building. Predicted (x axis) and experimentally measured (y axis) pIC₅₀ value correlations are shown. Values denote predictive capacity, goodness of fit, and, for the combined training and test sets, slope through the origin. E, graphical representation of the 3D-QSAR model from D. Gray shading, allowable space component of the model. Areas of steric constraints, steric freedom, electro-activity, and hydrophobic interactions are shown. Electrostatically active regions of the compound are shown in red and blue.

Figure 5.

3D-QSAR-informed optimization of select PK properties of the scaffold. A, metabolic stability predictions for the AVG-158 analog using SMARTCyp. Areas of high postulated metabolic susceptibility (red circles) against eight isoforms of cytochrome P450 are shown. Potential metabolic liabilities are color-coded from moderate (blue) to severe (orange). B, virtual library screen to optimize metabolic properties. Shown are predictions for six experimentally tested compounds (pink), virtual library entries (gray), and virtual library entries selected for synthesis (black). Dotted lines, cutoff criteria for chemical synthesis (predicted pIC₅₀ and metabolic stability at least equal to that of the least potent (predicted pIC₅₀ > 6.8) and most stable (1/PK score >0.147) experimentally analyzed analog, respectively). C, correlation of bioactivities predicted by the 3D-QSAR model (x axis) and experimentally measured (y axis) for the synthesized eight virtual screening hits selected in B. D, correlation between experimentally measured human microsome stability (x axis) and predicted metabolic susceptibility (y axis) for these eight virtual screening hits. The second-generation lead candidate AVG-233 is highlighted in C and D.

Figure 6.

PK profiling and mechanistic assessment of the AVG-233 lead. A, metabolic stability predictions for AVG-233 using SMARTCyp. B, single-dose PK testing of AVG-233 in mice. Animals (three per group) were dosed with AVG-233 intravenously (IV) or orally (PO) at the indicated dose levels. Plasma samples were prepared at the indicated time points and analyzed by LC/MS/MS. C, bioactivity testing of AVG-233 against RSV (red y axis) and RSV–, MeV–, or IAV-WSN–derived minigenome assays (blue y axis). Where applicable, four-parameter variable slope regression models and active concentrations are shown. D, in vitro RSV RdRp activity assay as described in Fig. 4C. The lead compound AVG-233 blocks 3′ RNA extension elongation but does not interfere with 3′ RNA extension by up to three nucleotides after de novo initiation from the promoter or back-priming. IAV-WSN RdRp values in C show means of six biological repeats. Symbols in B–D represent individual biological repeats (n = 3 each); error bars, S.D. IAV-WSN RdRp activity in C was determined in six biological repeats.

Figure 7.

Cytotoxicity profiling of AVG-233. A, assessment of cell metabolic activity after 48-h exposure of primary HBTECs to AVG-233. Assays were conducted in the presence of standard media, or RPMI supplemented with glucose or galactose as carbohydrate source to suppress glycolysis-induced masking of mitochondrial toxicity. B, multiplexed mitochondrial ToxGlo assay to determine cell membrane integrity and mitochondrial function after HBTEC exposure to AVG-233. C, in-cell ELISA determining effects on mitochondrial biogenesis after 48-h exposure to AVG-233. Symbols in A–C represent individual biological repeats (n = 3 each or greater); error bars, S.D.

Figure 8.

Antiviral potency of AVG-233. A, virus yield reduction of RSV A2-L19F and two RSV clinical strains by AVG-233. Infected cells were incubated in the presence of compound, and progeny virus titers were determined 48 h after infection. Active concentrations were determined through four-parameter variable slope regression modeling. LoD, level of detection. B, antiviral activity of first- and second-generation lead analogs AVG-158 and AVG-233, respectively, in undifferentiated primary HBTECs. Four-parameter variable slope regression models and active concentrations are shown. Symbols in A and B represent individual biological repeats (n = 3 each or greater); error bars, S.D.