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

• Published in: Nature chemical biology Vol. 12; no. 5; pp. 361 - 366
• Main Authors: Deans, Richard M, Morgens, David W, Ökesli, Ayşe, Pillay, Sirika, Horlbeck, Max A, Kampmann, Martin, Gilbert, Luke A, Li, Amy, Mateo, Roberto, Smith, Mark, Glenn, Jeffrey S, Carette, Jan E, Khosla, Chaitan, Bassik, Michael C
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.05.2016; Nature Publishing Group
• Subjects: 49/40; 631/154; 631/326/596; 631/92/507; 631/92/555; Antiviral Agents - pharmacology; Biochemical Engineering; Biochemistry; Bioorganic Chemistry; Biosynthesis; Carbazoles - chemistry; Carbazoles - pharmacology; Cell Biology; Cell Line, Tumor; Chemistry; Chemistry/Food Science; Cloning, Molecular; CRISPR-Cas Systems - genetics; Cytotoxicity; Dehydrogenase; Dengue virus; Drug resistance; Drugs; Humans; Lentivirus - drug effects; Lentivirus - physiology; Ribonucleic acid; RNA; RNA, Small Interfering - genetics; Vector-borne diseases; Viruses
• ISSN: 1552-4450; 1552-4469
• DOI: 10.1038/nchembio.2050

A combination of shRNA- and CRISPR-Cas9-based gene editing screens, corroborated by a metabolite suppression experiment identifies the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH) as the target of the broad-spectrum antiviral compound GSK983. 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.