Article | Published:

Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification

Nature Chemical Biology volume 12, pages 361366 (2016) | Download Citation

Abstract

Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and limitations of each screening method for identifying drug targets, and demonstrate the utility of parallel knockdown and knockout screens for comprehensive probing of drug activity.

  • Compound

    (R)-N-(6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-yl)picolinamide

  • Compound

    (R)-N-(6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-yl)-4-fluorobenzamide

  • Compound

    (R)-N-(6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-yl)-2-methylbenzamide

  • Compound

    (S)-N-(6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-yl)picolinamide

  • Compound

    (E)-2-(2-(4-chlorophenyl)hydrazono)cyclohexan-1-one

  • Compound

    6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-one

  • Compound

    (R)-6-chloro-N-((R)-1-(4-methoxyphenyl)ethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride

  • Compound

    (S)-6-chloro-N-((S)-1-(4-methoxyphenyl)ethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride

  • Compound

    (E)-2-(2-(4-bromophenyl)hydrazono)cyclohexan-1-one

  • Compound

    6-bromo-2,3,4,9-tetrahydro-1H-carbazol-1-one

  • Compound

    (R)-6-bromo-N-((R)-1-(4-methoxyphenyl)ethyl)-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride

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References

  1. 1.

    , & Current progress in antiviral strategies. Trends Pharmacol. Sci. 35, 86–102 (2014).

  2. 2.

    , & Intervention strategies for emerging viruses: use of antivirals. Curr. Opin. Virol. 3, 217–224 (2013).

  3. 3.

    et al. GSK983: a novel compound with broad-spectrum antiviral activity. Antiviral Res. 82, 1–11 (2009).

  4. 4.

    et al. Development of a broad-spectrum antiviral with activity against Ebola virus. Antiviral Res. 83, 245–251 (2009).

  5. 5.

    et al. Identification of an antioxidant small-molecule with broad-spectrum antiviral activity. Antiviral Res. 93, 23–29 (2012).

  6. 6.

    et al. FGI-104: a broad-spectrum small molecule inhibitor of viral infection. Am. J. Transl. Res. 1, 87–98 (2009).

  7. 7.

    et al. Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc. Natl. Acad. Sci. USA 101, 793–798 (2004).

  8. 8.

    et al. Identifying mechanism-of-action targets for drugs and probes. Proc. Natl. Acad. Sci. USA 109, 11178–11183 (2012).

  9. 9.

    et al. The Connectivity Map: using gene-expression signatures to connect small molecules, genes, and disease. Science 313, 1929–1935 (2006).

  10. 10.

    et al. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell 126, 611–625 (2006).

  11. 11.

    et al. Rethinking molecular similarity: comparing compounds on the basis of biological activity. ACS Chem. Biol. 7, 1399–1409 (2012).

  12. 12.

    & High-throughput RNAi screening in cultured cells: a user's guide. Nat. Rev. Genet. 7, 373–384 (2006).

  13. 13.

    , , & Target identification and mechanism of action in chemical biology and drug discovery. Nat. Chem. Biol. 9, 232–240 (2013).

  14. 14.

    et al. A systematic mammalian genetic interaction map reveals pathways underlying ricin susceptibility. Cell 152, 909–922 (2013).

  15. 15.

    et al. Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat. Methods 6, 443–445 (2009).

  16. 16.

    , & Integrated platform for genome-wide screening and construction of high-density genetic interaction maps in mammalian cells. Proc. Natl. Acad. Sci. USA 110, E2317–E2326 (2013).

  17. 17.

    , & Functional genomics platform for pooled screening and generation of mammalian genetic interaction maps. Nat. Protoc. 9, 1825–1847 (2014).

  18. 18.

    et al. Next-generation NAMPT inhibitors identified by sequential high-throughput phenotypic chemical and functional genomic screens. Chem. Biol. 20, 1352–1363 (2013).

  19. 19.

    et al. Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response. eLife 4, e07314 (2015).

  20. 20.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).

  21. 21.

    et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).

  22. 22.

    et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).

  23. 23.

    et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).

  24. 24.

    et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).

  25. 25.

    et al. Genome-scale CRISPR-Cas9 knockout screening in human cells. Science 343, 84–87 (2014).

  26. 26.

    , , & Genetic screens in human cells using the CRISPR-Cas9 system. Science 343, 80–84 (2014).

  27. 27.

    et al. Efficient asymmetric synthesis of N-[(1R)-6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-yl]-2-pyridinecarboxamide for treatment of human papillomavirus infections. Org. Process Res. Dev. 11, 539–545 (2007).

  28. 28.

    et al. Tetrahydrocarbazole amides with potent activity against human papillomaviruses. Bioorg. Med. Chem. Lett. 19, 4110–4114 (2009).

  29. 29.

    et al. Next-generation libraries for robust RNA interference-based genome-wide screens. Proc. Natl. Acad. Sci. USA 112, E3384–E3391 (2015).

  30. 30.

    et al. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system. Cell 155, 1479–1491 (2013).

  31. 31.

    et al. A tumor suppressor complex with GAP activity for the Rag GTPases that signal amino acid sufficiency to mTORC1. Science 340, 1100–1106 (2013).

  32. 32.

    & Regulation of mTORC1 by amino acids. Trends Cell Biol. 24, 400–406 (2014).

  33. 33.

    , , , & Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents. Structure 8, 25–33 (2000).

  34. 34.

    , , & Stimulation of de novo pyrimidine synthesis by growth signaling through mTOR and S6K1. Science 339, 1323–1328 (2013).

  35. 35.

    et al. Quantitative phosphoproteomics reveal mTORC1 activates de novo pyrimidine synthesis. Science 339, 1320–1323 (2013).

  36. 36.

    et al. Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool. BMC Bioinformatics 14, 128 (2013).

  37. 37.

    , , , & The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry 35, 1270–1273 (1996).

  38. 38.

    , , , & Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis. Proc. Natl. Acad. Sci. USA 108, 5777–5782 (2011).

  39. 39.

    et al. Inhibition of dengue virus through suppression of host pyrimidine biosynthesis. J. Virol. 85, 6548–6556 (2011).

  40. 40.

    et al. Identification of broad-spectrum antiviral compounds and assessment of the druggability of their target for efficacy against respiratory syncytial virus (RSV). Proc. Natl. Acad. Sci. USA 108, 6739–6744 (2011).

  41. 41.

    et al. Mechanism of action for leflunomide in rheumatoid arthritis. Clin. Immunol. 93, 198–208 (1999).

  42. 42.

    , , & Importance of ribonucleotide availability to proliferating T-lymphocytes from healthy humans. Disproportionate expansion of pyrimidine pools and contrasting effects of de novo synthesis inhibitors. J. Biol. Chem. 270, 29682–29689 (1995).

  43. 43.

    et al. Characterization of dengue virus resistance to brequinar in cell culture. Antimicrob. Agents Chemother. 54, 3686–3695 (2010).

  44. 44.

    et al. The global distribution and burden of dengue. Nature 496, 504–507 (2013).

  45. 45.

    , , & Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase. A versatile tool to rate inhibitor effects? Eur. J. Biochem. 268, 1861–1868 (2001).

  46. 46.

    , , & Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase. Protein Expr. Purif. 13, 414–422 (1998).

  47. 47.

    et al. Dengue virus capsid protein usurps lipid droplets for viral particle formation. PLoS Pathog. 5, e1000632 (2009).

  48. 48.

    et al. Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53. Virology 230, 300–308 (1997).

  49. 49.

    , , , & Interplay of acute and persistent infections caused by Venezuelan equine encephalitis virus encoding mutated capsid protein. J. Virol. 84, 10004–10015 (2010).

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Acknowledgements

We thank J. Weissman, O. Chen, K. Han, B. Lowry, J. Kuo, N. Plugis and K. Nguyen for helpful discussions, and A. Brunet, J. Sage and D. Vollrath for critical reading of this manuscript. M.K. was supported by NIH/NCI K99/R00 CA181494. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE-114747 (R.M.D.). R.M.D. was additionally supported by a Burt and DeeDee McMurtry Stanford Graduate Fellowship. This work was funded by US National Institues of Health grants U19-AI109662 and Director's New Innovator Award Program 1DP2HD084069-01, and a seed grant from Stanford ChEM-H. Any opinion, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

Author information

Affiliations

  1. Department of Chemistry, Stanford University, Stanford, California, USA.

    • Richard M Deans
    • , Ayşe Ökesli
    •  & Chaitan Khosla
  2. Department of Genetics, Stanford University, Stanford, California, USA.

    • Richard M Deans
    • , David W Morgens
    • , Amy Li
    •  & Michael C Bassik
  3. Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.

    • Sirika Pillay
    • , Roberto Mateo
    • , Jeffrey S Glenn
    •  & Jan E Carette
  4. Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research and Howard Hughes Medical Institute, San Francisco, California, USA.

    • Max A Horlbeck
    • , Martin Kampmann
    •  & Luke A Gilbert
  5. Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.

    • Mark Smith
    • , Jeffrey S Glenn
    • , Jan E Carette
    • , Chaitan Khosla
    •  & Michael C Bassik
  6. Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, California, USA.

    • Jeffrey S Glenn
  7. Department of Chemical Engineering, Stanford University, Stanford, California, USA.

    • Chaitan Khosla
  8. Department of Biochemistry, Stanford University, Stanford, California, USA.

    • Chaitan Khosla

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Contributions

R.M.D. synthesized and characterized GSK983 and analogs, performed the genome-wide shRNA and CRISPR-Cas9 screens, assisted with statistical analysis of genomic screen results, validated genomic screen hits, designed and conducted GSK983 biological activity assays, pyrimidine supplementation experiments, and cell-cycle analyses, and assisted with DENV and VEEV antiviral assays. D.W.M. designed the maximum likelihood estimator and conducted statistical analysis of genomic screen results. A.Ö. expressed and purified DHODH and performed in vitro enzymatic assays. R.M.D. and A.Ö. expressed and purified CMPK1, and performed in vitro enzymatic assays. S.P. assisted with DENV and VEEV antiviral assays. J.E.C. and R.M. constructed the DENV luciferase reporter. M.K. designed the shRNA library with assistance from M.A.H. and M.C.B. M.A.H. designed the CRISPR-Cas9 sgRNA library with assistance from L.A.G. and M.C.B. A.L. provided technical support. J.E.C. provided guidance for DENV antiviral assays. M.S., J.S.G., C.K. and M.C.B. conceived of the study. R.M.D., C.K. and M.C.B. wrote the manuscript and prepared the figures.

Competing interests

Stanford University has filed a patent application based on the findings in this report.

Corresponding authors

Correspondence to Chaitan Khosla or Michael C Bassik.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–4.

  2. 2.

    Supplementary Note

    Synthetic Procedures

Excel files

  1. 1.

    Supplementary Data Set 1

    Complete gene rankings from the genome-wide shRNA screen

  2. 2.

    Supplementary Data Set 2

    Complete gene rankings from the genome-wide CRISPRCas9 screen

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DOI

https://doi.org/10.1038/nchembio.2050

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