scholarly journals The cellular response to drug perturbation is limited: comparison of large-scale chemogenomic fitness signatures

2021 ◽  
Author(s):  
Marjan Barazandeh ◽  
Divya Kriti ◽  
Corey Nislow ◽  
Guri Giaever
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Cellular Response ◽  
Large Scale ◽  
Genetic Interaction ◽  
Common Mechanism ◽  
Genome Wide ◽  
Powerful Approach ◽  
Response Systems ◽  
Academic Laboratory

Abstract BackgroundChemogenomic profiling is a powerful approach towards understanding the genome-wide cellular response to small molecules. Developed in Saccharomyces cerevisiae, chemogenomic screens provide direct, unbiased identification of drug target candidates as well as genes required for drug resistance. While many laboratories have performed chemogenomic fitness assays, they have not been assessed for reproducibility and accuracy. Here we analyze the two largest independent yeast chemogenomic datasets comprising over 35 million gene-drug interactions and more than 6000 unique chemogenomic profiles; the first from our own academic laboratory and the second from the Novartis Institute of Biomedical Research (NIBR).ResultsCombining the datasets revealed robust genetic interaction response signatures that point to common mechanism of action, despite the substantial differences in experimental and analytical pipelines. We previously reported that the cellular response to small molecules is limited and can be described by a network of 45 chemogenomic signatures. In the present study, we show that the majority of these signatures (66%) are also found in the companion dataset, providing further support for their biological relevance as systems-level, small molecule response systems. ConclusionsOur results demonstrate the robustness of chemogenomic fitness profiling in yeast, while offering guidelines for performing other high-dimensional comparisons including parallel CRISPR screens in mammalian cells.

scholarly journals Efficient strategies for screening large-scale genetic interaction networks

2017 ◽  
Author(s):  
Raamesh Deshpande ◽  
Justin Nelson ◽  
Scott W. Simpkins ◽  
Michael Costanzo ◽  
Jeff S. Piotrowski ◽  
...  
Keyword(s):  
Large Scale ◽  
Optimal Algorithm ◽  
Genetic Interaction ◽  
Genetic Screens ◽  
Chemical Genetic ◽  
Genome Wide ◽  
Powerful Approach ◽  
Interaction Screening ◽  
Genetic Interaction Data

Large-scale genetic interaction screening is a powerful approach for unbiased characterization of gene function and understanding systems-level cellular organization. While genome-wide screens are desirable as they provide the most comprehensive interaction profiles, they are resource and time-intensive and sometimes infeasible, depending on the species and experimental platform. For these scenarios, optimal methods for more efficient screening while still producing the maximal amount of information from the resulting profiles are of interest.To address this problem, we developed an optimal algorithm, called COMPRESS-GI, which selects a small but informative set of genes that captures most of the functional information contained within genome-wide genetic interaction profiles. The utility of this algorithm is demonstrated through an application of the approach to define a diagnostic mutant set for large-scale chemical genetic screens, where more than 13,000 compound screens were achieved through the increased throughput enabled by the approach. COMPRESS-GI can be broadly applied for directing genetic interaction screens in other contexts, including in species with little or no prior genetic-interaction data.

scholarly journals Interrogation of kinase genetic interactions provides a global view of PAK1-mediated signal transduction pathways

2020 ◽  
Vol 295 (50) ◽  
pp. 16906-16919
Author(s):  
Jae-Hong Kim ◽  
Yeojin Seo ◽  
Myungjin Jo ◽  
Hyejin Jeon ◽  
Young-Seop Kim ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Cell Migration ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Drug Targets ◽  
Large Scale ◽  
Genetic Interaction ◽  
Genetic Interactions ◽  
Signal Transduction Pathways ◽  
Global View

Kinases are critical components of intracellular signaling pathways and have been extensively investigated with regard to their roles in cancer. p21-activated kinase-1 (PAK1) is a serine/threonine kinase that has been previously implicated in numerous biological processes, such as cell migration, cell cycle progression, cell motility, invasion, and angiogenesis, in glioma and other cancers. However, the signaling network linked to PAK1 is not fully defined. We previously reported a large-scale yeast genetic interaction screen using toxicity as a readout to identify candidate PAK1 genetic interactions. En masse transformation of the PAK1 gene into 4,653 homozygous diploid Saccharomyces cerevisiae yeast deletion mutants identified ∼400 candidates that suppressed yeast toxicity. Here we selected 19 candidate PAK1 genetic interactions that had human orthologs and were expressed in glioma for further examination in mammalian cells, brain slice cultures, and orthotopic glioma models. RNAi and pharmacological inhibition of potential PAK1 interactors confirmed that DPP4, KIF11, mTOR, PKM2, SGPP1, TTK, and YWHAE regulate PAK1-induced cell migration and revealed the importance of genes related to the mitotic spindle, proteolysis, autophagy, and metabolism in PAK1-mediated glioma cell migration, drug resistance, and proliferation. AKT1 was further identified as a downstream mediator of the PAK1-TTK genetic interaction. Taken together, these data provide a global view of PAK1-mediated signal transduction pathways and point to potential new drug targets for glioma therapy.

scholarly journals Dynamics of Uptake and Metabolism of Small Molecules in Cellular Response Systems

PLoS ONE ◽  
2009 ◽  
Vol 4 (3) ◽  
pp. e4923 ◽  
Author(s):  
Maria Werner ◽  
Szabolcs Semsey ◽  
Kim Sneppen ◽  
Sandeep Krishna
Keyword(s):  
Small Molecules ◽  
Cellular Response ◽  
Response Systems ◽  
Uptake And Metabolism

scholarly journals A one-step tRNA-CRISPR system for genome-wide genetic interaction mapping in mammalian cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yulei Zhao ◽  
Kathrin Tyrishkin ◽  
Calvin Sjaarda ◽  
Prem Khanal ◽  
Jeff Stafford ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Drug Targets ◽  
Genetic Interaction ◽  
Genetic Interactions ◽  
Cancer Treatments ◽  
Genome Wide ◽  
A Genome ◽  
High Efficient ◽  
One Step ◽  
Screening Accuracy

Abstract Mapping genetic interactions in mammalian cells is limited due to technical obstacles. Here we describe a method called TCGI (tRNA-CRISPR for genetic interactions) to generate a high-efficient, barcode-free and scalable pairwise CRISPR libraries in mammalian cells for identifying genetic interactions. We have generated a genome- wide library to identify genes genetically interacting with TAZ in cell viability regulation. Validation of candidate synergistic genes reveals the screening accuracy of 85% and TAZ-MCL1 is characterized as combinational drug targets for non-small cell lung cancer treatments. TCGI has dramatically improved the current methods for mapping genetic interactions and screening drug targets for combinational therapies.

scholarly journals Multiplex base editing to convert TAG into TAA codons in the human genome

2021 ◽  
Author(s):  
Yuting Chen ◽  
Eriona Hysolli ◽  
Anlu Chen ◽  
Stephen Casper ◽  
Songlei Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Genome ◽  
Mammalian Cells ◽  
Large Scale ◽  
Essential Gene ◽  
Human Cells ◽  
Viral Resistance ◽  
Computational Tool ◽  
Base Editing ◽  
Genome Wide

Large-scale recoding has been shown to enable novel amino acids, biocontainment and viral resistance in bacteria only so far. Here we extend this to human cells demonstrating exceptional base editing to convert TAG to TAA for 33 essential genes via a single transfection, and examine base-editing genome-wide (observing ~ 40 C-to-T off-target events in essential gene exons). We also introduce GRIT, a computational tool for recoding. This demonstrates the feasibility of recoding, and multiplex editing in mammalian cells.

scholarly journals High-throughput small molecule screening reveals Nrf2-dependent and -independent pathways of cellular stress resistance

2020 ◽  
Vol 6 (40) ◽  
pp. eaaz7628
Author(s):  
David B. Lombard ◽  
William J. Kohler ◽  
Angela H. Guo ◽  
Christi Gendron ◽  
Melissa Han ◽  
...  
Keyword(s):  
Small Molecules ◽  
Stress Resistance ◽  
Mammalian Cells ◽  
Large Scale ◽  
Cellular Stress ◽  
Methyl Methanesulfonate ◽  
Genetic Studies ◽  
Resistance Induction ◽  
Small Molecule Screening ◽  
Evolutionarily Conserved

Aging is the dominant risk factor for most chronic diseases. Development of antiaging interventions offers the promise of preventing many such illnesses simultaneously. Cellular stress resistance is an evolutionarily conserved feature of longevity. Here, we identify compounds that induced resistance to the superoxide generator paraquat (PQ), the heavy metal cadmium (Cd), and the DNA alkylator methyl methanesulfonate (MMS). Some rescue compounds conferred resistance to a single stressor, while others provoked multiplex resistance. Induction of stress resistance in fibroblasts was predictive of longevity extension in a published large-scale longevity screen in Caenorhabditis elegans, although not in testing performed in worms and flies with a more restricted set of compounds. Transcriptomic analysis and genetic studies implicated Nrf2/SKN-1 signaling in stress resistance provided by two protective compounds, cardamonin and AEG 3482. Small molecules identified in this work may represent attractive tools to elucidate mechanisms of stress resistance in mammalian cells.

scholarly journals Author Correction: A one-step tRNA-CRISPR system for genome-wide genetic interaction mapping in mammalian cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yulei Zhao ◽  
Kathrin Tyrishkin ◽  
Calvin Sjaarda ◽  
Prem Khanal ◽  
Jeff Stafford ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genetic Interaction ◽  
Crispr System ◽  
Genome Wide ◽  
One Step

scholarly journals Genetic screens in isogenic mammalian cell lines without single cell cloning

2019 ◽  
Author(s):  
Peter C DeWeirdt ◽  
Kendall R Sanson ◽  
Ruth E Hanna ◽  
Mudra Hegde ◽  
Annabel K Sangree ◽  
...  
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Genetic Interaction ◽  
Cell Types ◽  
Repair Gene ◽  
Synthetic Lethal ◽  
Mammalian Cell Lines ◽  
Genome Wide ◽  
Single Cell Cloning ◽  
Synthetic Lethal Interactions

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.

scholarly journals Cellular response to small molecules that selectively stall protein synthesis by the ribosome

2018 ◽  
Author(s):  
Nadège Liaud ◽  
Max A. Horlbeck ◽  
Luke A. Gilbert ◽  
Ketrin Gjoni ◽  
Jonathan S. Weissman ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Synthesis ◽  
Small Molecules ◽  
Cellular Response ◽  
Genetic Interaction ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Genetic Modifiers ◽  
A Genome ◽  
Therapeutic Development

ABSTRACTIdentifying small molecules that inhibit protein synthesis by selectively stalling the ribosome constitutes a new strategy for therapeutic development. Compounds that inhibit the translation of PCSK9, a major regulator of low-density lipoprotein cholesterol, have been identified that reduce LDL cholesterol in preclinical models and that affect the translation of only a few off-target proteins. Although some of these compounds hold potential for future therapeutic development, it is not known how they impact the physiology of cells or ribosome quality control pathways. Here we used a genome-wide CRISPRi screen to identify proteins and pathways that modulate cell growth in the presence of high doses of a selective PCSK9 translational inhibitor, PF-06378503 (PF8503). The two most potent genetic modifiers of cell fitness in the presence of PF8503, the ubiquitin binding protein ASCC2 and helicase ASCC3, bind to the ribosome and protect cells from toxic effects of high concentrations of the compound. Surprisingly, translation quality control proteins Pelota (PELO) and HBS1L sensitize cells to PF8503 treatment. In genetic interaction experiments, ASCC3 acts together with ASCC2, and functions downstream of HBS1L. Taken together, these results identify new connections between ribosome quality control pathways, and provide new insights into the selectivity of compounds that stall human translation that will aid the development of next-generation selective translation stalling compounds to treat disease.

scholarly journals Identifying Biomolecular Targets of the Anticancer Vitamin-E-δ-Tocotrienol Using a Computational Approach: Virtual Target Screening

2020 ◽  
pp. 1-8
Author(s):  
Wesley H. Brooks ◽  
Yuri Pevzner ◽  
Elza Pevzner ◽  
Kenyon G. Daniel ◽  
Wayne C. Guida ◽  
...  
Keyword(s):  
Vitamin E ◽  
Small Molecules ◽  
Cellular Response ◽  
Large Scale ◽  
Initial Step ◽  
Antitumor Agent ◽  
Computational Approach ◽  
Virtual Target ◽  
Target Screening ◽  
Virtual Target Screening

In recent years, evidence has mounted that a particular form of vitamin E (its δ-tocotrienol variant) may have cellular functions beyond that of an antioxidant, a role commonly ascribed to the tocotrienol class of compounds. In particular, numerous studies of δ-tocotrienol’s effect on cancer cells have identified it as a potent anticancer and antitumor agent. However, this important revelation of potential therapeutic use poses a series of new challenges, with arguably the most important being the elucidation of the precise mechanism of action responsible for the anticancer activity of δ-tocotrienol. As an initial step to address this question, we have used a computational tool, Virtual Target Screening (a molecular docking-based tool that identifies potential binding partners for small molecules), to identify potential biomolecular targets of δ-tocotrienol. Then, to gain a consensus as to the type of biomolecular entity that could be a target for δ-tocotrienol, we utilized PharmMapper and PASS (a ligand-based chemoinformatic approach), and ProBiS (a tool that analyses binding site similarities across known proteins). The results of our multipronged computational consensus-seeking approach showed that such a strategy can identify potential cellular targets of small molecules. This is evidenced by our identification of estrogen receptor-beta, a protein that has been previously shown to bind δ-tocotrienol, which elicited a cellular response. This study supports the use of such a computational approach as an initial step in target identification to avoid time-consuming, costly large-scale experimental screening, greatly reducing the experimental work to just one or a few candidate proteins.

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