Exploring the Mode-of-Action of Bioactive Compounds by Chemical-Genetic Profiling in Yeast

Parsons, Ainslie B.; López, Andrés Aguilera; Givoni, Inmar E.; Williams, David E.; Gray, Christopher A.; Porter, Justin; Chua, Gordon; Sopko, Richelle; Brost, Renée L.; Ho, Cheuk-Hei; Wang, Jiyi; Ketela, Troy; Brenner, Charles; Brill, Julie A.; Fernández, G. Esteban; Lorenz, Todd C.; Payne, Grégory S.; Ishihara, Satoru; Ohya, Yoshikazu; Andrews, Brenda; Hughes, Timothy R.; Frey, Brendan J.; Graham, Todd R.; Andersen, Raymond J.; Boone, Charles

doi:10.1016/j.cell.2006.06.040

Cited by 466 publications

(525 citation statements)

References 67 publications

(83 reference statements)

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“…The subsequent generation of a phenotypic miniarray profile (P-MAP) does not require the construction of an exhaustive set of double mutants and is therefore less labor intensive than E-MAP techniques and more readily applied to other systems (e.g., RNA interference in human cells). In principle, a P-MAP could be constructed using available phenotypic data from large-scale drug sensitivity studies (Parsons et al, 2006). However, we find biochemical readouts of phenotypes relevant to trafficking processes-such as CPY or pro-alpha factor secretion, or cell surface chitin synthesis-lead to the identification of a greater number of complexes, with more significant p values, than growth-based assays (unpublished observations).…”

Section: Discussionmentioning

confidence: 99%

“…This set is expected to include most components of endosomal transport pathways and complexes, with the exception of those with essential or redundant functions. Because genes that act together can often be identified based on shared mutant phenotypes (Schuldiner et al, 2005;Parsons et al, 2006;Quenneville and Conibear, 2006), we reasoned that quantitative phenotypic profiling might resolve these genes into functional groupings that represent individual complexes or transport steps. We rearrayed the selected 279 mutants and 93 randomly chosen controls to create a "miniarray" that reduces experimental variation by allowing phenotypes to be evaluated on a single plate.…”

Section: Identification Of Functionally Related Genes By Phenotypic Mmentioning

confidence: 99%

“…Yeast knockout screens that record fitness defects under conditions of chemical, environmental, or genetic perturbation have successfully identified the target pathways of bioactive compounds and increased our understanding of genetic redundancy Parsons et al, 2006;St. Onge et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

Schluter

Lam

Brumm

et al. 2008

MBoC

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Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. INTRODUCTIONEndosomal protein sorting is a highly conserved cellular process that is required for receptor down-regulation, viral replication, and development (Katzmann et al., 2002;Morita and Sundquist, 2004). Much progress has been made in recent years in identifying distinct multiprotein complexes that regulate the fusion of primary endocytic vesicles, the maturation of an early endosome into a multivesicular body (MVB), and the recycling of proteins and lipids to other organelles (Gruenberg and Stenmark, 2004;Bonifacino and Rojas, 2006). The ESCRT-I, -II, and -III complexes (endosomal-sorting complex required for transport), which act in sequence to regulate the formation of internal vesicles at the MVB, play a central role in endosome biogenesis (Katzmann et al., 2002;Hurley and Emr, 2006). Other transport complexes regulate the targeting and fusion of endosomes or endosome-derived vesicles with other organelles (Bowers and Stevens, 2005;Bonifacino and Rojas, 2006).Many components of the endosomal-sorting machinery were first identified in yeast genetic screens for mutants that are defective in protein transport to the yeast vacuole, which requires functional endosomal sorting and recycling (Bowers and Stevens, 2005). Most of these components have a conserved role in endosome biogenesis in higher cells. Mammalian homologues have been identified for most if not all yeast ESCRT subunits (von Schwedler et al., 2003;Hurley and Emr, 2006), and at least some of the machinery that regulates recycling from the MVB is also well conserved. For example, the five-subunit retromer complex that mediates endosome-to-trans-Golgi network (TGN) transport in yeast is associated with tubular regions of the endosome in mammalian cells and mediates retrograde transport (Arighi et al., 2004;Seaman, 2004). In the past 20 years, classical genetic screens have identified more than 50 vacuole protein-sorting (VPS) genes (Bowers and Stevens, 2005). Surprisingly, ...

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Section: Discussionmentioning

confidence: 99%

Section: Identification Of Functionally Related Genes By Phenotypic Mmentioning

confidence: 99%

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Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

Schluter

Lam

Brumm

et al. 2008

MBoC

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“…In signature-based approaches, a series of drug-induced molecular/phenotypic measurements are made in an experimental system. Collections of measurements from many small molecules form multivariate signatures that aim to fingerprint drugs based on their relative signature similarity (8)(9)(10)(11)(12)(13). In several landmark studies using Saccharomyces cerevisiae; gene expression compendia (8); and, later, barcoded loss of function/ORF libraries (9,10), large signatures were shown to characterize individual small molecule mechanisms of action effectively.…”

mentioning

confidence: 99%

Defining principles of combination drug mechanisms of action

Pritchard¹,

Bruno²,

Gilbert³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

154

137

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Combination chemotherapies have been a mainstay in the treatment of disseminated malignancies for almost 60 y, yet even successful regimens fail to cure many patients. Although their singledrug components are well studied, the mechanisms by which drugs work together in clinical combination regimens are poorly understood. Here, we combine RNAi-based functional signatures with complementary informatics tools to examine drug combinations. This approach seeks to bring to combination therapy what the knowledge of biochemical targets has brought to single-drug therapy and creates a statistical and experimental definition of "combination drug mechanisms of action." We show that certain synergistic drug combinations may act as a more potent version of a single drug. Conversely, unlike these highly synergistic combinations, most drugs average extant single-drug variations in therapeutic response. When combined to form multidrug regimens, averaging combinations form averaging regimens that homogenize genetic variation in mouse models of cancer and in clinical genomics datasets. We suggest surprisingly simple and predictable combination mechanisms of action that are independent of biochemical mechanism and have implications for biomarker discovery as well as for the development of regimens with defined genetic dependencies.chemotherapy | lymphoma | RNAi signature | systems biology C urrent rationales for the design of combination chemotherapy regimens were developed in the 1940s and 1950s (1-5) and, remarkably, were concurrent with the identification of DNA as the genetic material. The development of these regimens in the absence of any knowledge of cancer genomics was a remarkable achievement. However, although our knowledge of the genetic drivers of cancer and the mechanisms of drug action has increased dramatically over the past 30 y, this information has been difficult to adapt to clinical practice. As such, even very successful combination regimens often fail to cure many patients (6, 7). We hypothesize that part of this failure is due to the absence of mechanistic information about how drugs in regimens interact to promote combination effects (we term these effects "combination mechanisms of action"). We sought to address this gap by investigating specific hypotheses concerning the "mechanisms of action" of combination therapy.The classic term "drug mechanism of action" refers to the description of a specific biochemical event, which is often the activation or inhibition of an enzymatic effect. However, in recent years, "signature"-based prediction has provided a powerful new strategy for examining drug mechanism. In signature-based approaches, a series of drug-induced molecular/phenotypic measurements are made in an experimental system. Collections of measurements from many small molecules form multivariate signatures that aim to fingerprint drugs based on their relative signature similarity (8-13). In several landmark studies using Saccharomyces cerevisiae; gene expression compendia (8); and, later, barcoded loss o...

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“…We refer to synthetic lethal interactions between a chemical compound and a genetic mutation as ''composite synthetic lethality'' (CSL). CSL has been used as a method to identify potential targets of bioactive compounds in yeast (8)(9)(10)(11)(12). Likewise, CSL should also be effective in identifying novel bioactive compounds that inhibit a particular pathway.…”

mentioning

confidence: 99%

Composite synthetic lethal identification of membrane traffic inhibitors

Duncan¹,

Huang

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Small molecule inhibitors provide powerful tools to characterize highly dynamic and complex eukaryotic cell pathways such as those mediating membrane traffic. However, a lack of easy and generalizable assays has constrained identification of novel inhibitors despite availability of diverse chemical libraries. Here, we report a facile growth-based strategy in yeast to screen for pathway-specific inhibitors. The approach uses well characterized synthetic genetic growth defects to guide design of cells genetically sensitized for inhibition of chosen pathways. With this strategy, we identified a family of piperazinyl phenylethanone compounds as inhibitors of traffic between the trans-Golgi network (TGN) and endosomes that depends on the clathrin adaptor complex AP-1. The compounds did not significantly alter other trafficking pathways involving the TGN or endosomes, indicating specificity. Compound treatment also altered localization of AP-1 in mammalian cells. These previously uncharacterized inhibitors will be useful for future studies of clathrin-mediated transport in yeast, and potentially in other organisms. Furthermore, the easily automated technology should be adaptable for identification of inhibitors of other cellular processes.clathrin ͉ small molecule ͉ AP-1 ͉ GGA ͉ yeast

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Exploring the Mode-of-Action of Bioactive Compounds by Chemical-Genetic Profiling in Yeast

Cited by 466 publications

References 67 publications

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

Defining principles of combination drug mechanisms of action

Composite synthetic lethal identification of membrane traffic inhibitors

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