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Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase

Nature Microbiology volume 5, pages 1232–1246 (2020)Cite this article

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Abstract

Paramyxoviruses such as human parainfluenza virus type-3 (HPIV3) and measles virus (MeV) are a substantial health threat. In a high-throughput screen for inhibitors of HPIV3 (a major cause of acute respiratory infection), we identified GHP-88309—a non-nucleoside inhibitor of viral polymerase activity that possesses unusual broad-spectrum activity against diverse paramyxoviruses including respiroviruses (that is, HPIV1 and HPIV3) and morbilliviruses (that is, MeV). Resistance profiles of distinct target viruses overlapped spatially, revealing a conserved binding site in the central cavity of the viral polymerase (L) protein that was validated by photoaffinity labelling-based target mapping. Mechanistic characterization through viral RNA profiling and in vitro MeV polymerase assays identified a block in the initiation phase of the viral polymerase. GHP-88309 showed nanomolar potency against HPIV3 isolates in well-differentiated human airway organoid cultures, was well tolerated (selectivity index > 7,111) and orally bioavailable, and provided complete protection against lethal infection in a Sendai virus mouse surrogate model of human HPIV3 disease when administered therapeutically 48 h after infection. Recoverees had acquired robust immunoprotection against reinfection, and viral resistance coincided with severe attenuation. This study provides proof of the feasibility of a well-behaved broad-spectrum allosteric antiviral and describes a chemotype with high therapeutic potential that addresses major obstacles of anti-paramyxovirus drug development.

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Fig. 1: Identification and initial characterization of GHP-88309.
Fig. 2: GHP-88309 targets a conserved microdomain in the L protein, inhibiting de novo RNA synthesis.
Fig. 3: GHP-88309 is efficacious in 3D-ALI-HBTEC.
Fig. 4: Pharmacokinetics characterization of GHP-88309.
Fig. 5: GHP-88309 is efficacious in an SeV mouse surrogate assay of HPIV infection.
Fig. 6: Effect of GHP-88309 treatment on adaptive immunity and rechallenge, and correlation between resistance and viral pathogenesis.

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Data availability

Confocal microscopy and histology raw data files have been deposited at Figshare (access links are specified in the figure captions). HPIV3 whole-genome sequencing data of the DMSO- and GHP-88309-treated passages are available from the NCBI BioProject (PRJNA561835). SeV NGS data are available from the NCBI Gene Expression Omnibus database (GSE140376). HTS raw data are available from the corresponding author upon request. However, no chemical structure information concerning the composition of screening libraries and unsuccessful hit candidates will be provided. Source data are provided with this paper.

Code availability

The open-source MScreen package is available from the Center for Chemical Genomics at the University of Michigan. The LAVA package is available at https://github.com/michellejlin/lava. The chemical filter algorithms used are available at http://swissadme.ch and http://pasilla.health.unm.edu/tomcat/badapple/badapple. Homology modelling software is available from the Swiss homology modelling server at http://swissmodel.expasy.org. The power analysis software used is available at http://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower.html. All other software solutions employed are commercially available from the developing houses.

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Acknowledgements

We thank M. T. Saindaine and M. A. Lockwood for chemical synthesis, J. Wolf for assistance with molecular biology, H.-Y. Tang and the Wistar Institute Proteomics and Metabolomics Facility for assistance with proteomics analysis, B. R. tenOever for NGS support, K. K. Conzelmann for the BSR-T7/5 stable cell line and R. T. Jacob for IT support. The MScreen software package was kindly provided by the Center for Chemical Genomics of the University of Michigan under a licence agreement by the University of Michigan Office of Technology Transfer; JChem was used for structure database management, search and prediction (JChem 6.2; ChemAxon (2014)); and Marvin was employed for drawing, displaying and characterizing chemical structures, substructures and reactions (Marvin 14.9.22.0; ChemAxon (2014)). This work was supported in part by Public Health Service grants AI071002 (to R.K.P.) and HD079327 (to R.K.P.), from the NIH/NIAID and NIH/NICHD, respectively. The funders had no role in study design, data collection and interpretation or the decision to submit the work for publication.

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Authors and Affiliations

  1. Institute for Biomedical Sciences, Georgia State University, Atlanta, GA, USA

    Robert M. Cox, Julien Sourimant, Mart Toots, Jeong-Joong Yoon & Richard K. Plemper

  2. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Satoshi Ikegame, Ruth E. Watkinson, Patricia Thibault & Benhur Lee

  3. Emory Institute for Drug Development, Emory University, Atlanta, GA, USA

    Mugunthan Govindarajan, Jose R. Marengo, Zachary Sticher, Alexander A. Kolykhalov & Michael G. Natchus

  4. Virology Division, Department of Laboratory Medicine, University of Washington, Seattle, WA, USA

    Negar Makhsous, Michelle J. Lin & Alexander L. Greninger

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Contributions

R.M.C. performed and analysed most of the experiments. J.S. performed the RdRP assays. M.T. and J.-J.Y. assisted with the animal and organoid studies. S.I., R.E.W., P.T., N.M., M.J.L., A.L.G. and B.L. performed the NGS experiments and data analysis. M.G., J.R.M., Z.S., A.A.K. and M.G.N. performed the chemical synthesis and pharmacokinetics analyses. R.K.P. contributed to the HTS and biochemical experiments, and supervised the study.

Corresponding author

Correspondence to Richard K. Plemper.

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Competing interests

R.M.C., B.L. and R.K.P. are co-inventors on a provisional patent application ‘2-fluoro-6-isoquinolin-5-ylbenzamide, and antiviral uses related thereto’, covering the method of use of GHP-88309 for paramyxovirus disease therapy. This study could affect their personal financial statuses. All other authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Anti-HPIV3 HTS hit identification.

Anti-HPIV3 HTS hit identification. a, Schematic of genome organization of recHPIV3-JS-NanoLuc. b, Validation of automated HTS protocol in 384-well format, using recHPIV3-JS-NanoLuc as detection agent. Three 384-well assay validation plates featuring alternating columns containing vehicle (Max) or the broad-spectrum host-directed inhibitor JMN3-003 in intermediate (0.5 × EC50; Med) or sterilizing (10 × EC50; Min) concentrations. On each validation plate, control columns were arranged in different order. Results for one plate are shown, performance parameters for all three plates and statistical assessments are provided in Supplementary Table 8. c, Dose-response antiviral activity and cytotoxicity tests with sourced hit candidates GHP-88309 and GHP-64627. d, Comparison of antiviral activity of commercially sourced and resynthesized GHP-88309. No significant differences were noted (P=0.7723). Statistical analysis through the extra sum-of-squares F test. e, Effect of GHP-88309 on cell proliferation of different immortalized cell lines. No toxicity was detectable in the concentration range tested (up to 100 µM). f, Effect of GHP-88309 on metabolic activity of primary human BTECs. Cytotoxicity was assessed by measuring COX-1 protein levels relative to vehicle treated HBTECs. Effects of compound incubation was assessed in a dose-response format with the highest concentration equal to 1000 µM. Mitochondrial toxicity was tested in parallel after by measuring SDH-A protein levels relative to vehicle treated HBTECs. Protein levels were measured after 72-hour incubation with GHP-88309. g, MeV infected cells (m.o.i. 0.5 TCID50 units/cell) were incubated with GHP-88309 at increasing compound concentrations for 48 hours. Cell viability was measured and normalized to uninfected, vehicle treated cells using PrestoBlue assay. GHP-88309 exhibited no toxicity at concentrations up 300 µM. In (c–g), symbols represent biological repeats, lines are derived from 4-parameter variable slope regression modeling (c–g). Where applicable, active (EC50) and cytotoxic (CC50) concentrations are shown with 95% confidence intervals (CIs) in parentheses.

Source data

Extended Data Fig. 2 GHP-88309 hit validation.

GHP-88309 hit validation. a, Antiviral activity of GHP-88309 determined under different conditions (after infection at high m.o.i.; at different media pH; in the presence of BSA). No significant differences were noted (P=0.329). Statistical analysis through the extra sum-of-squares F test. Symbols represent biological repeats, lines connect data means. b-d, Efficacy of GHP-88309 against different clinical isolates of HPIV3 (10L3 (KY973583), 9R4 (KY674929), 3-1, 3-2, and 3-3) (b), MeV (c), and HPIV1 (4C5, 2D4 (MF554715.1), 5M6 (MF554714.1), and 5F6) (d). Progeny virus yields from dose-response tests were determined through TCID50 titration. GHP-88309 consistently inhibited all target virus strains with low-micromolar to submicromolar potency; EC50 concentrations are shown. e, HPIV3 ToA studies. GHP-88309 was added at the specified time points after infection at 20 µM. The host-directed polymerase inhibitor JMN3-003 was included for reference.

Source data

Extended Data Fig. 3 Characterization of viral resistance mutations to GHP-88309.

Characterization of viral resistance mutations to GHP-88309. a, All candidate resistance mutations emerging from adaptation of HPIV3 and two engineered combinations thereof were rebuilt and tested in an HPIV minigenome assay of RdRP activity in the presence of GHP-88309. b, Mutations from (a) that were rebuilt in recHPIV3-JS-NanoLuc and tested against GHP-88309 in reporter dose-response assays. c, Candidate mutations emerging from MeV adaptation were rebuilt and tested in an MeV minigenome assay of RdRP activity. d, All candidate resistance mutations emerging from SeV adaptation were rebuilt in recSeV and tested against the resulting recSeVs in virus yield-based dose-response assays. In (a-d), symbols represent biological repeats, lines are derived from 4-parameter variable slope regression modeling, and active concentrations (EC50 and EC90 if applicable) are shown with 95% CIs. Yellow highlights denote experimentally confirmed resistance mutations.

Source data

Extended Data Fig. 4 GHP-88309-016 target mapping through photoaffinity labeling.

GHP-88309-016 target mapping through photoaffinity labeling. a, Structure of GHP-88309-016. b, GHP-88309-016 is bioactive, potently inhibiting MeV and HPIV3 replication without appreciable cytotoxicity (n=3). EC50 values from 4-parameter variable slope regression models are shown with 95% CIs. c, SDS-PAGE fractionation of purified MeV L1708 constructs used in UV crosslinking and BLI studies on 7.5% gels, followed by Coomassie blue staining. Polymerase complexes were purified once in sufficient quantity to meet the needs of the project. d, 2D-schematic of the MeV L protein with locations of known resistance mutations (top) and peptides identified by photoaffinity labeling (bottom; black bars). Cyan, green, yellow, orange, and red depict the RdRP, capping, connector, MTase, and C-terminal domains. e, Homology model of MeV L showing the locations of the peptides crosslinked to GHP-88309-016 (black spheres). Confirmed GHP-88309 resistance sites are shown in red. Peptides 1 and 2 are located on the exterior of the polymerase. f, Only residues of peptide 3 (black) are exposed to the interior channels of the polymerase in proximity of the resistance sites (red). The homology model was based on the coordinates reported for HPIV5 L (PDB: 6v85).

Source data

Extended Data Fig. 5 In silico docking of GHP-88309.

In silico docking of GHP-88309. a, Ribbon representation of the MeV L internal channel, showing distances between confirmed resistance sites (red) and the nearest residue in photoaffinity labeled peptide 3 (D993; black). Polymerase domain color-coding as in Fig. 2a. b, Docking of GHP-88309 (blue sticks) and GHP-88309-016 (yellow sticks) into the MeV L model, using D993 and confirmed resistance hot-spots as target site guides. The top-scoring pose is conserved between GHP-88309 and GHP-88309-016, and positions the between capping and RdRP domains. The aryl-azide moiety is located approximately 7.8 Å from residue D993 in peptide 3. Numbers denote predicted free energy associated with this docking pose. c, 2D-diagram of predicted top-scoring ligand interaction generated with MOE. Predicted are hydrogen bond interactions between the isoquinoline ring of GHP-88309 and residues Y942 and R865. The benzamide moiety is posited between residues Q1007 and R1011, thus overall linking RdRP and capping domains. d, e, Application of the equivalent in silico docking approach as in (b) to NiV (d) and RSV L (e). Top scoring poses in NiV (pink sticks) and RSV (white sticks) L are distinct from that in MeV L (superimposed as blue sticks). Known resistance mutations are colored red (a-b, d, e). NiV homology model is based on HPIV5 L (PDB: 6v85); RSV L is native (PDB: 6pzk).

Extended Data Fig. 6 Steady-state analyses of BLI binding saturation and sensitization of NiV L to GHP-88309.

Steady-state analyses of BLI binding saturation and sensitization of NiV L to GHP-88309. a, Concentration-dependent steady-state BLI sensor response signals were plotted for the different MeV L1708 samples (standard (WT) or carrying resistance mutations as indicated) to probe whether saturation of binding was reached. b, NiV minigenome assays to test inhibitory activity of GHP-88309 against standard NiV RdRP and NiV RdRP harboring an LH1165Y point mutation. Symbols represent biological repeats, lines are derived from 4-parameter variable slope regression modeling, and active concentrations (EC50 and EC90 if applicable) are shown with 95% CIs.

Source data

Extended Data Fig. 7 Effect of GHP-88309 treatment on HPIV3 and MeV primary transcription.

Effect of GHP-88309 treatment on HPIV3 and MeV primary transcription. a-c, GHP-88309 significantly reduced levels of MeV P (a), H (b), and L (c) mRNA transcripts at 12 hours after infection. d, GHP-88309 significantly reduced the synthesis of HPIV3 Le RNA 2 hours after infection. e, f, GHP-88309 did not significantly alter the HPIV3 (e; 2 hours after infection) and MeV (f; 4 hours after infection) primary mRNA transcription gradients. Values represent relative changes compared to vehicle treated samples (a-d) or relative changes compared to HPIV3 N (e) or MeV P (f) mRNA levels. Experiments were conducted in at least three biological repeats, determined in duplicate each. In (a–f), symbols represent individual biological repeats, columns denote means, and error bars represent SD. Statistical significance was determined by one-way ANOVA (a–d) or two-way ANOVA (e, f) using Dunnett’s multiple comparison post-hoc test (two-sided).

Source data

Extended Data Fig. 8 GHP-88309 inhibits de novo initiation of RNA synthesis at the promoter.

GHP-88309 inhibits de novo initiation of RNA synthesis at the promoter. a, b, RSV-derived RNA template (a) that was used in Fig. 2j and is capable of back-priming depicted in (b). c, Repeat result of the MeV RdRP assay with the RSV-derived template shown in Fig. 2; originals are provided in Source Data file (Plemper_SourceData_Fig2_Originals_Repeats.pdf). d, MeV-derived RNA template, incapable of back-priming. e, MeV RdRP assay with the MeV-derived template shown (d). The experiment was performed three times with similar results. Originals and repeats are provided in (Plemper_SourceData_ED_Fig8_Originals_Repeats.pdf). Higher molecular weight material in (e) likely represents poly-adenylation consistent with previous results obtained with a similar template and NiV polymerase complexes48.

Source data

Extended Data Fig. 9 Effect of GHP-88309 treatment on host innate immune response activation.

Effect of GHP-88309 treatment on host innate immune response activation. Individual measurement of data summarized in Fig. 5h are shown. a, Relative expression of genes involved in innate host antiviral response pathways, determined through qRT-PCR three, six, and nine days after SeV infection of mice. Results are shown for vehicle treated (black), prophylactically treated (2 hours before infection; red), and therapeutically treated (48 hours after infection; blue) mice, all normalized to mock-infected and untreated reference animals (n=3 in all groups). Statistical analysis with two-way ANOVA and Tukey’s multiple comparison post-hoc test. b, Relative induction of type-1 IFN and interferon stimulated genes in undifferentiated HBTECs after HPIV3 infection and treatment with 6 µM GHP-88309. Cells were mock infected and exposed to GHP-88309 (treated mock inf), HPIV3 infected and vehicle-treated (vehicle), or HPIV3 infected and GHP-88309 treated starting 2 hours before infection. All results are expressed relative to untreated, mock-infected cells. Symbols represent measurements from individual mice (a) or individual biological repeats (b), columns in (b) show data means (n=3) throughout, error bars represent SD. Statistical analysis with one-way ANOVA and Tukey’s multiple comparison post-hoc test; NS, not significant.

Source data

Extended Data Fig. 10 Mapping of RSV L capping blocker and AZ-27 resistance hotspots.

Mapping of RSV L capping blocker and AZ-27 resistance hotspots. Analogous positions of resistance mutations against the RSV L capping inhibitor compound C (E1269D and I1381S; magenta spheres) and initiation blocker AZ-27 (Y1631; orange spheres) are projected onto an HPIV3 L homology model when locatable. Resistance mutations against the capping inhibitor are proximal to conserved PRNTase motifs (blue spheres) and distal to GHP-88309 resistance mutations (red spheres). The RdRP, connector, and capping domains are color-coded as in Fig. 2a. The HPIV3 L homology model is based on the coordinates reported for HPIV5 L (PDB: 6v85).

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, source data to Supplementary Fig. 2 and Supplementary Tables 1–8.

Reporting Summary

Supplementary Computational Data 1

DMSO-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage DMSO-1 after growth in the presence of DMSO over ten passages. Passage 1 (PIV3-1-1-DMSO); passage 5 (PIV3-5-1-DMSO); and passage 10 (PIV3-10-1-DMSO).

Supplementary Computational Data 2

DMSO-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage DMSO-2 after growth in the presence of DMSO over ten passages. Passage 1 (PIV3-1-2-DMSO); passage 5 (PIV3-5-2-DMSO); and passage 10 (PIV3-10-2-DMSO).

Supplementary Computational Data 3

DMSO-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage DMSO-3 after growth in the presence of DMSO over ten passages. Passage 1 (PIV3-1-3-DMSO); passage 5 (PIV3-5-3-DMSO); and passage 10 (PIV3-10-3-DMSO).

Supplementary Computational Data 4

DMSO-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage DMSO-4 after growth in the presence of DMSO over ten passages. Passage 1 (PIV3-1-4-DMSO); passage 5 (PIV3-5-4-DMSO); and passage 10 (PIV3-10-4-DMSO).

Supplementary Computational Data 5

DMSO-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage DMSO-5 after growth in the presence of DMSO over ten passages. Passage 1 (PIV3-1-5-DMSO); passage 5 (PIV3-5-5-DMSO); and passage 10 (PIV3-10-5-DMSO).

Supplementary Computational Data 6

DMSO-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage DMSO-6 after growth in the presence of DMSO over ten passages. Passage 1 (PIV3-1-6-DMSO); passage 5 (PIV3-5-6-DMSO); and passage 10 (PIV3-10-6-DMSO).

Supplementary Computational Data 7

Drug-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage Drug-1 after growth in the presence of increasing GHP-88309 concentrations over ten passages. Passage 1 (PIV3-1-1-05DRUG) = 0.5 µM; passage 5 (PIV3-5-1-3DRUG) = 3 µM; and passage 10 (PIV3-10-1-50DRUG) = 50 µM.

Supplementary Computational Data 8

Drug-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage Drug-2 after growth in the presence of increasing GHP-88309 concentrations over ten passages. Passage 1 (PIV3-1-2-05DRUG) = 0.5 µM; passage 5 (PIV3-5-2-3DRUG) = 3 µM; and passage 10 (PIV3-10-2-50DRUG) = 50 µM.

Supplementary Computational Data 9

Drug-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage Drug-3 after growth in the presence of increasing GHP-88309 concentrations over ten passages. Passage 1 (PIV3-1-3-05DRUG) = 0.5 µM; passage 5 (PIV3-5-3-3DRUG) = 3 µM; and passage 10 (PIV3-10-3-50DRUG) = 50 µM.

Supplementary Computational Data 10

Drug-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage Drug-4 after growth in the presence of increasing GHP-88309 concentrations over ten passages. Passage 1 (PIV3-1-4-05DRUG) = 0.5 µM; passage 5 (PIV3-5-4-3DRUG) = 3 µM; and passage 10 (PIV3-10-4-50DRUG) = 50 µM.

Supplementary Computational Data 11

Drug-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage Drug-5 after growth in the presence of increasing GHP-88309 concentrations over ten passages. Passage 1 (PIV3-1-5-05DRUG) = 0.5 µM; passage 5 (PIV3-5-5-3DRUG) = 3 µM; and passage 10 (PIV3-10-5-50DRUG) = 50 µM.

Supplementary Computational Data 12

Drug-treated HPIV3 NGS analysis. Whole-genome NGS of HPIV3-JS lineage Drug-6 after growth in the presence of increasing GHP-88309 concentrations over ten passages. Passage 1 (PIV3-1-6-05DRUG) = 0.5 µM; passage 5 (PIV3-5-6-3DRUG) = 3 µM; and passage 10 (PIV3-10-6-50DRUG) = 50 µM.

Supplementary Computational Data 13

SeV L NGS analysis for SeV lineage 1 after 13 passages in increasing concentrations of GHP-88309 (Drug-1; 200 µM at passage 13) or DMSO (DMSO-1).

Supplementary Computational Data 14

SeV L NGS analysis for SeV lineage 2 after 13 passages in increasing concentrations of GHP-88309 (Drug-2; 200 µM at passage 13) or DMSO (DMSO-2).

Supplementary Computational Data 15

SeV L NGS analysis for SeV lineage 3 after 13 passages in increasing concentrations of GHP-88309 (Drug-3; 200 µM at passage 13) or DMSO (DMSO-3).

Supplementary Computational Data 16

SeV L NGS analysis for SeV lineage 4 after 13 passages in increasing concentrations of GHP-88309 (Drug-4; 200 µM at passage 13) or DMSO (DMSO-4).

Supplementary Computational Data 17

SeV L NGS analysis for SeV lineage 5 after 13 passages in increasing concentrations of GHP-88309 (Drug-5; 200 µM at passage 13) or DMSO (DMSO-5).

Supplementary Computational Data 18

SeV L NGS analysis for SeV lineage 6 after 13 passages in increasing concentrations of GHP-88309 (Drug-6; 200 µM at passage 13) or DMSO (DMSO-6).

Source data

Source Data Fig. 1

Quantitative source data.

Source Data Fig. 2

Quantitative source data and statistical source data.

Source Data Fig. 2

Repeat of BLI experiments, unprocessed autoradiograms and repeat experiment autoradiograms.

Source Data Fig. 3

Quantitative source data.

Source Data Fig. 4

Quantitative source data and statistical source data.

Source Data Fig. 5

Quantitative source data and statistical source data.

Source Data Fig. 6

Quantitative source data and statistical source data.

Source Data Extended Data Fig. 1

Quantitative source data and statistical source data.

Source Data Extended Data Fig. 2

Quantitative source data and statistical source data.

Source Data Extended Data Fig. 3

Quantitative source data.

Source Data Extended Data Fig. 4

Quantitative source data.

Source Data Extended Data Fig. 4

Unprocessed gel.

Source Data Extended Data Fig. 6

Quantitative source data.

Source Data Extended Data Fig. 7

Quantitative source data and statistical source data.

Source Data Extended Data Fig. 8

Unprocessed autoradiograms and repeat experiment autoradiograms.

Source Data Extended Data Fig. 9

Quantitative source data and statistical source data.

Supplementary Dataset 1

Source data to Supplementary Table 3.

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Cox, R.M., Sourimant, J., Toots, M. et al. Orally efficacious broad-spectrum allosteric inhibitor of paramyxovirus polymerase. Nat Microbiol 5, 1232–1246 (2020). https://doi.org/10.1038/s41564-020-0752-7

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