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Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs

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 gen...

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Autores principales: Kittanakom, Saranya, Arnoldo, Anthony, Brown, Kevin R., Wallace, Iain, Kunavisarut, Tada, Torti, Dax, Heisler, Lawrence E., Surendra, Anuradha, Moffat, Jason, Giaever, Guri, Nislow, Corey
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737177/
https://www.ncbi.nlm.nih.gov/pubmed/23797109
http://dx.doi.org/10.1534/g3.113.006437
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author Kittanakom, Saranya
Arnoldo, Anthony
Brown, Kevin R.
Wallace, Iain
Kunavisarut, Tada
Torti, Dax
Heisler, Lawrence E.
Surendra, Anuradha
Moffat, Jason
Giaever, Guri
Nislow, Corey
author_facet Kittanakom, Saranya
Arnoldo, Anthony
Brown, Kevin R.
Wallace, Iain
Kunavisarut, Tada
Torti, Dax
Heisler, Lawrence E.
Surendra, Anuradha
Moffat, Jason
Giaever, Guri
Nislow, Corey
author_sort Kittanakom, Saranya
collection PubMed
description 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-Abl−positive 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 by more 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 drug−target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug−target 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.
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spelling pubmed-37371772013-08-08 Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs Kittanakom, Saranya Arnoldo, Anthony Brown, Kevin R. Wallace, Iain Kunavisarut, Tada Torti, Dax Heisler, Lawrence E. Surendra, Anuradha Moffat, Jason Giaever, Guri Nislow, Corey G3 (Bethesda) Investigations 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-Abl−positive 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 by more 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 drug−target pairs, we successfully identify 40% of the targets described in the literature and uncover several unanticipated drug−target 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. Genetics Society of America 2013-08-01 /pmc/articles/PMC3737177/ /pubmed/23797109 http://dx.doi.org/10.1534/g3.113.006437 Text en Copyright © 2013 Kittanakom et al. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Kittanakom, Saranya
Arnoldo, Anthony
Brown, Kevin R.
Wallace, Iain
Kunavisarut, Tada
Torti, Dax
Heisler, Lawrence E.
Surendra, Anuradha
Moffat, Jason
Giaever, Guri
Nislow, Corey
Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs
title Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs
title_full Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs
title_fullStr Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs
title_full_unstemmed Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs
title_short Miniature Short Hairpin RNA Screens to Characterize Antiproliferative Drugs
title_sort miniature short hairpin rna screens to characterize antiproliferative drugs
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737177/
https://www.ncbi.nlm.nih.gov/pubmed/23797109
http://dx.doi.org/10.1534/g3.113.006437
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