Publication: Miniature short hairpin rna screens to characterize antiproliferative drugs
Issued Date
2013-01-01
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ISSN
21601836
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2-s2.0-84883212102
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Mahidol University
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SCOPUS
Bibliographic Citation
G3: Genes, Genomes, Genetics. Vol.3, No.8 (2013), 1375-1387
Suggested Citation
Saranya Kittanakom, Anthony Arnoldo, Kevin R. Brown, Iain Wallace, Tada Kunavisarut, Dax Torti, Lawrence E. Heisler, Anuradha Surendra, Jason Moffat, Guri Giaever, Corey Nislow Miniature short hairpin rna screens to characterize antiproliferative drugs. G3: Genes, Genomes, Genetics. Vol.3, No.8 (2013), 1375-1387. doi:10.1534/g3.113.006437 Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/31425
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Title
Miniature short hairpin rna screens to characterize antiproliferative drugs
Abstract
The application of new proteomics and genomics technologies support a view in which few drugs act solely by inhibiting a single cellular target. Indeed, drug activity is modulated by complex, often incompletely understood cellular mechanisms. Therefore, efforts to decipher mode of action through genetic perturbation such as RNAi typically yields "hits" that fall into several categories. Of particular interest to the present study, we aimed to characterize secondary activities of drugs on cells. Inhibiting a known target can result in clinically relevant synthetic phenotypes. In one scenario, drug perturbation could, for example, improperly activate a protein that normally inhibits a particular kinase. In other cases, additional, lower affinity targets can be inhibited as in the example of inhibition of c-Kit observed in Bcr-Abl2positive cells treated with Gleevec. Drug transport and metabolism also play an important role in the way any chemicals act within the cells. Finally, RNAi per se can also affect cell fitness bymore general off-target effects, e.g., via the modulation of apoptosis or DNA damage repair. Regardless of the root cause of these unwanted effects, understanding the scope of a drug's activity and polypharmacology is essential for better understanding its mechanism(s) of action, and such information can guide development of improved therapies. We describe a rapid, cost-effective approach to characterize primary and secondary effects of small-molecules by using small-scale libraries of virally integrated short hairpin RNAs. We demonstrate this principle using a "minipool" composed of shRNAs that target the genes encoding the reported protein targets of approved drugs. Among the 28 known reported drug2target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug2target interactions based on drug-induced synthetic lethality. We provide a detailed protocol for performing such screens and for analyzing the data. This cost-effective approach to mammalian knockdown screens, combined with the increasing maturation of RNAi technology will expand the accessibility of similar approaches in academic settings. © 2013 Kittanakom et al.