title: 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.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California, USA.|Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Deans
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: David W
authors.initials: DW
authors.lastname: Morgens
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California, USA.
authors.forename: Ayşe
authors.initials: A
authors.lastname: Ökesli
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.
authors.forename: Sirika
authors.initials: S
authors.lastname: Pillay
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research and Howard Hughes Medical Institute, San Francisco, California, USA.
authors.forename: Max A
authors.initials: MA
authors.lastname: Horlbeck
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research and Howard Hughes Medical Institute, San Francisco, California, USA.
authors.forename: Martin
authors.identifier: http://orcid.org/0000-0002-3819-7019
authors.initials: M
authors.lastname: Kampmann
authors.affiliation: Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biomedical Research and Howard Hughes Medical Institute, San Francisco, California, USA.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Gilbert
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Li
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Mateo
authors.affiliation: Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Smith
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.|Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.|Division of Gastroenterology and Hepatology, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Glenn
authors.affiliation: Department of Microbiology and Immunology, Stanford University, Stanford, California, USA.|Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.
authors.forename: Jan E
authors.initials: JE
authors.lastname: Carette
authors.affiliation: Department of Chemistry, Stanford University, Stanford, California, USA.|Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.|Department of Chemical Engineering, Stanford University, Stanford, California, USA.|Department of Biochemistry, Stanford University, Stanford, California, USA.
authors.forename: Chaitan
authors.initials: C
authors.lastname: Khosla
authors.affiliation: Department of Genetics, Stanford University, Stanford, California, USA.|Stanford University Chemistry, Engineering, and Medicine for Human Health (ChEM-H), Stanford, California, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Bassik
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002227:Carbazoles / Q000737:chemistry / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006801:Humans; D016086:Lentivirus / Q000187:drug effects* / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics*
chemical_list: D000998:Antiviral Agents; D002227:Carbazoles; C541365:N-((1R)-6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-yl)-2-pyridinecarboxamide; D034741:RNA, Small Interfering