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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.

No MeSH data available.


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GSK983 inhibits DHODH to block virus replication and cell proliferation. (a) Schematic representation of mammalian pyrimidine metabolism. Genes that appeared as strong sensitizing hits in the shRNA screen (CMPK1, DHODH, UCK2) and CRISPR-Cas9 screen (UCK2) are highlighted in yellow. (b) Dihydroorotic acid had no effect on GSK983-induced growth inhibition in K562 cells. (c) Orotic acid reversed GSK983-induced growth inhibition in K562 cells. For (b) and (c), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of (dihydro)orotic acid. Error bars represent ± standard deviation of 4 biological replicates. (d) GSK983 and analogues inhibited recombinant human DHODH in vitro. Ki values are averages of two independent Ki determinations at different inhibitor concentrations. The range between independently calculated Ki values for each inhibitor is shown in parentheses. IC50 values for inhibition of episomal HPV-16 replication in cell-based antiviral assays are those reported by GlaxoSmithKline3. (e) Structures of GSK983, 6Br-pF, 6Br-oTol, and GSK984.
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Figure 2: GSK983 inhibits DHODH to block virus replication and cell proliferation. (a) Schematic representation of mammalian pyrimidine metabolism. Genes that appeared as strong sensitizing hits in the shRNA screen (CMPK1, DHODH, UCK2) and CRISPR-Cas9 screen (UCK2) are highlighted in yellow. (b) Dihydroorotic acid had no effect on GSK983-induced growth inhibition in K562 cells. (c) Orotic acid reversed GSK983-induced growth inhibition in K562 cells. For (b) and (c), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of (dihydro)orotic acid. Error bars represent ± standard deviation of 4 biological replicates. (d) GSK983 and analogues inhibited recombinant human DHODH in vitro. Ki values are averages of two independent Ki determinations at different inhibitor concentrations. The range between independently calculated Ki values for each inhibitor is shown in parentheses. IC50 values for inhibition of episomal HPV-16 replication in cell-based antiviral assays are those reported by GlaxoSmithKline3. (e) Structures of GSK983, 6Br-pF, 6Br-oTol, and GSK984.

Mentions: While the CRISPR-Cas9 and shRNA screens each highlighted unique hit genes and together provided a more complete understanding of the biological activity of GSK983, we considered that the highly sensitizing hits in the pyrimidine biosynthesis pathway were among the most likely candidates to be molecular targets of GSK983. We reasoned that cells expressing an shRNA against a protein target of GSK983 should be highly sensitized to GSK983-induced growth inhibition. Consequently, we focused our target identification effort on the pyrimidine metabolism genes DHODH and CMPK1, which were the top sensitizing hits from our genome-wide shRNA screen. Mammalian cells derive pyrimidine (deoxy)ribonucleotide triphosphates either from de novo biosynthesis or pyrimidine salvage, in which intact pyrimidine metabolites are recycled from intracellular nucleic acid degradation or imported into the cell from exogenous sources (Fig. 2a). DHODH (dihydroorotate dehydrogenase) is required for de novo pyrimidine biosynthesis, while CMPK1 plays a critical role in both de novo biosynthesis and pyrimidine salvage (Fig. 2a).


Parallel shRNA and CRISPR-Cas9 screens enable antiviral drug target identification
GSK983 inhibits DHODH to block virus replication and cell proliferation. (a) Schematic representation of mammalian pyrimidine metabolism. Genes that appeared as strong sensitizing hits in the shRNA screen (CMPK1, DHODH, UCK2) and CRISPR-Cas9 screen (UCK2) are highlighted in yellow. (b) Dihydroorotic acid had no effect on GSK983-induced growth inhibition in K562 cells. (c) Orotic acid reversed GSK983-induced growth inhibition in K562 cells. For (b) and (c), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of (dihydro)orotic acid. Error bars represent ± standard deviation of 4 biological replicates. (d) GSK983 and analogues inhibited recombinant human DHODH in vitro. Ki values are averages of two independent Ki determinations at different inhibitor concentrations. The range between independently calculated Ki values for each inhibitor is shown in parentheses. IC50 values for inhibition of episomal HPV-16 replication in cell-based antiviral assays are those reported by GlaxoSmithKline3. (e) Structures of GSK983, 6Br-pF, 6Br-oTol, and GSK984.
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Figure 2: GSK983 inhibits DHODH to block virus replication and cell proliferation. (a) Schematic representation of mammalian pyrimidine metabolism. Genes that appeared as strong sensitizing hits in the shRNA screen (CMPK1, DHODH, UCK2) and CRISPR-Cas9 screen (UCK2) are highlighted in yellow. (b) Dihydroorotic acid had no effect on GSK983-induced growth inhibition in K562 cells. (c) Orotic acid reversed GSK983-induced growth inhibition in K562 cells. For (b) and (c), viable cells were counted by flow cytometry (FSC/SSC) following 72 h treatment with 48 nM GSK983 or vehicle and the indicated concentration of (dihydro)orotic acid. Error bars represent ± standard deviation of 4 biological replicates. (d) GSK983 and analogues inhibited recombinant human DHODH in vitro. Ki values are averages of two independent Ki determinations at different inhibitor concentrations. The range between independently calculated Ki values for each inhibitor is shown in parentheses. IC50 values for inhibition of episomal HPV-16 replication in cell-based antiviral assays are those reported by GlaxoSmithKline3. (e) Structures of GSK983, 6Br-pF, 6Br-oTol, and GSK984.
Mentions: While the CRISPR-Cas9 and shRNA screens each highlighted unique hit genes and together provided a more complete understanding of the biological activity of GSK983, we considered that the highly sensitizing hits in the pyrimidine biosynthesis pathway were among the most likely candidates to be molecular targets of GSK983. We reasoned that cells expressing an shRNA against a protein target of GSK983 should be highly sensitized to GSK983-induced growth inhibition. Consequently, we focused our target identification effort on the pyrimidine metabolism genes DHODH and CMPK1, which were the top sensitizing hits from our genome-wide shRNA screen. Mammalian cells derive pyrimidine (deoxy)ribonucleotide triphosphates either from de novo biosynthesis or pyrimidine salvage, in which intact pyrimidine metabolites are recycled from intracellular nucleic acid degradation or imported into the cell from exogenous sources (Fig. 2a). DHODH (dihydroorotate dehydrogenase) is required for de novo pyrimidine biosynthesis, while CMPK1 plays a critical role in both de novo biosynthesis and pyrimidine salvage (Fig. 2a).

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