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

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.

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Figure 1: shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983.
Figure 2: GSK983 inhibits DHODH to block virus replication and cell proliferation.
Figure 3: Deoxycytidine reverses the antiproliferative effect of GSK983 but not antiviral activity.

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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.

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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.

Corresponding authors

Correspondence to Chaitan Khosla or Michael C Bassik.

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

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–7 and Supplementary Tables 1–4. (PDF 7399 kb)

Supplementary Data Set 1

Complete gene rankings from the genome-wide shRNA screen (XLSX 2220 kb)

Supplementary Data Set 2

Complete gene rankings from the genome-wide CRISPRCas9 screen (XLSX 1571 kb)

Supplementary Note

Synthetic Procedures (PDF 202 kb)

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Deans, R., Morgens, D., Ökesli, A. et al. Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification. Nat Chem Biol 12, 361–366 (2016). https://doi.org/10.1038/nchembio.2050

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