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

View Article: PubMed Central - PubMed

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 use parallel genome-wide high-coverage shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad spectrum antiviral with unexplained cytotoxicity1–3. We show that GSK983 blocks 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 reduces GSK983 cytotoxicity but not antiviral activity, providing an attractive novel approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Together, 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 comprehensively probing drug activity.

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shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983. (a) Structure of GSK983. (b) GSK983 dose response in K562 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h GSK983 treatment at the indicated concentration. Error bars represent ± standard deviation of 8 biological replicates from two independent experiments. Schematic representation of genome-wide shRNA (c) and CRISPR-Cas9 (d) screens. (e) Top ten hits from the shRNA and CRISPR-Cas9 screens in cellular and biological context. Circle size is proportional to MLE score absolute value. Square size is proportional to median fold-enrichment or disenrichment. (f) Comparative analysis of results from shRNA and CRISPR-Cas9 screens. Pyrimidine metabolism (orange), CoQ10 biosynthesis (blue), regulation of mTORC1 activity (green). (g) Validation of selected top hit genes from the shRNA screen using a competitive growth assay. A total of 27 shRNAs were retested (6 targeting DHODH; 4 targeting CMPK1; 3 each targeting COQ2, PDSS2, PDSS1, and COQ10B; and 5 negative controls). Error bars represent ± standard deviation of log(mCherry enrichment ratio) values for all retested shRNAs targeting each gene. P values were calculated by Mann-Whitney U test. Validation of selected sensitizing (h) or protective (i) sgRNAs from the CRISPR-Cas9 screen using a competitive growth assay. Bars represent the average of two biological replicates.
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Figure 1: shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983. (a) Structure of GSK983. (b) GSK983 dose response in K562 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h GSK983 treatment at the indicated concentration. Error bars represent ± standard deviation of 8 biological replicates from two independent experiments. Schematic representation of genome-wide shRNA (c) and CRISPR-Cas9 (d) screens. (e) Top ten hits from the shRNA and CRISPR-Cas9 screens in cellular and biological context. Circle size is proportional to MLE score absolute value. Square size is proportional to median fold-enrichment or disenrichment. (f) Comparative analysis of results from shRNA and CRISPR-Cas9 screens. Pyrimidine metabolism (orange), CoQ10 biosynthesis (blue), regulation of mTORC1 activity (green). (g) Validation of selected top hit genes from the shRNA screen using a competitive growth assay. A total of 27 shRNAs were retested (6 targeting DHODH; 4 targeting CMPK1; 3 each targeting COQ2, PDSS2, PDSS1, and COQ10B; and 5 negative controls). Error bars represent ± standard deviation of log(mCherry enrichment ratio) values for all retested shRNAs targeting each gene. P values were calculated by Mann-Whitney U test. Validation of selected sensitizing (h) or protective (i) sgRNAs from the CRISPR-Cas9 screen using a competitive growth assay. Bars represent the average of two biological replicates.

Mentions: We first examined the biological activity of GSK983 (Fig. 1a and Supplementary Results, Supplementary Fig. 1a) in human K562 cells. GSK983 inhibited K562 cell growth with an IC50 of 21 nM (Fig. 1b and Supplementary Fig. 1b), consistent with previous observations1. Cell cycle analysis revealed that 24 h GSK983 treatment caused an accumulation of K562 cells in S phase (Supplementary Fig. 1c,d), while prolonged 72 h treatment induced a dose-dependent increase in K562 cell death by apoptosis (Supplementary Fig. 1e,f).


Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983. (a) Structure of GSK983. (b) GSK983 dose response in K562 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h GSK983 treatment at the indicated concentration. Error bars represent ± standard deviation of 8 biological replicates from two independent experiments. Schematic representation of genome-wide shRNA (c) and CRISPR-Cas9 (d) screens. (e) Top ten hits from the shRNA and CRISPR-Cas9 screens in cellular and biological context. Circle size is proportional to MLE score absolute value. Square size is proportional to median fold-enrichment or disenrichment. (f) Comparative analysis of results from shRNA and CRISPR-Cas9 screens. Pyrimidine metabolism (orange), CoQ10 biosynthesis (blue), regulation of mTORC1 activity (green). (g) Validation of selected top hit genes from the shRNA screen using a competitive growth assay. A total of 27 shRNAs were retested (6 targeting DHODH; 4 targeting CMPK1; 3 each targeting COQ2, PDSS2, PDSS1, and COQ10B; and 5 negative controls). Error bars represent ± standard deviation of log(mCherry enrichment ratio) values for all retested shRNAs targeting each gene. P values were calculated by Mann-Whitney U test. Validation of selected sensitizing (h) or protective (i) sgRNAs from the CRISPR-Cas9 screen using a competitive growth assay. Bars represent the average of two biological replicates.
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Figure 1: shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983. (a) Structure of GSK983. (b) GSK983 dose response in K562 cells. Viable cells were counted by flow cytometry (FSC/SSC) following 72 h GSK983 treatment at the indicated concentration. Error bars represent ± standard deviation of 8 biological replicates from two independent experiments. Schematic representation of genome-wide shRNA (c) and CRISPR-Cas9 (d) screens. (e) Top ten hits from the shRNA and CRISPR-Cas9 screens in cellular and biological context. Circle size is proportional to MLE score absolute value. Square size is proportional to median fold-enrichment or disenrichment. (f) Comparative analysis of results from shRNA and CRISPR-Cas9 screens. Pyrimidine metabolism (orange), CoQ10 biosynthesis (blue), regulation of mTORC1 activity (green). (g) Validation of selected top hit genes from the shRNA screen using a competitive growth assay. A total of 27 shRNAs were retested (6 targeting DHODH; 4 targeting CMPK1; 3 each targeting COQ2, PDSS2, PDSS1, and COQ10B; and 5 negative controls). Error bars represent ± standard deviation of log(mCherry enrichment ratio) values for all retested shRNAs targeting each gene. P values were calculated by Mann-Whitney U test. Validation of selected sensitizing (h) or protective (i) sgRNAs from the CRISPR-Cas9 screen using a competitive growth assay. Bars represent the average of two biological replicates.
Mentions: We first examined the biological activity of GSK983 (Fig. 1a and Supplementary Results, Supplementary Fig. 1a) in human K562 cells. GSK983 inhibited K562 cell growth with an IC50 of 21 nM (Fig. 1b and Supplementary Fig. 1b), consistent with previous observations1. Cell cycle analysis revealed that 24 h GSK983 treatment caused an accumulation of K562 cells in S phase (Supplementary Fig. 1c,d), while prolonged 72 h treatment induced a dose-dependent increase in K562 cell death by apoptosis (Supplementary Fig. 1e,f).

View Article: PubMed Central - PubMed

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 use parallel genome-wide high-coverage shRNA and CRISPR-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad spectrum antiviral with unexplained cytotoxicity1–3. We show that GSK983 blocks 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 reduces GSK983 cytotoxicity but not antiviral activity, providing an attractive novel approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Together, 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 comprehensively probing drug activity.

No MeSH data available.


Related in: MedlinePlus