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Design, synthesis, and characterization of a highly effective Hog1 inhibitor: a powerful tool for analyzing MAP kinase signaling in yeast.

Dinér P, Veide Vilg J, Kjellén J, Migdal I, Andersson T, Gebbia M, Giaever G, Nislow C, Hohmann S, Wysocki R, Tamás MJ, Grøtli M - PLoS ONE (2011)

Bottom Line: These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo.Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress.Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

View Article: PubMed Central - PubMed

Affiliation: Medicinal Chemistry, Department of Chemistry, University of Gothenburg, Göteborg, Sweden.

ABSTRACT
The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

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In vitro Hog1 kinase activity assays.(A) Efficacy of compounds 1a–e,                                4a–e, and SB203580. (B)                                IC50 curves for compounds 4a,                                4b, and SB203580. Kinase assays were                            performed in a kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM                                MgCl2, 2 mM DTT) containing 0.4 µg GST-Hog1, 0.2 mM                            ATP, 0.1 µCi/nmol [32γP]ATP, and 100                            µM peptide substrate, and Hog1 activity was determined as                            described in the Experimental section. Kinase reactions were performed                            in the presence of 0.1 µM inhibitor (A) or with a range of                            inhibitor concentrations (B). The concentration of the DMSO vehicle was                            identical in all reactions (1% final). The results are the                            average of three independent experiments and the error bars represent                            the standard deviation (s.d.).
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pone-0020012-g005: In vitro Hog1 kinase activity assays.(A) Efficacy of compounds 1a–e, 4a–e, and SB203580. (B) IC50 curves for compounds 4a, 4b, and SB203580. Kinase assays were performed in a kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT) containing 0.4 µg GST-Hog1, 0.2 mM ATP, 0.1 µCi/nmol [32γP]ATP, and 100 µM peptide substrate, and Hog1 activity was determined as described in the Experimental section. Kinase reactions were performed in the presence of 0.1 µM inhibitor (A) or with a range of inhibitor concentrations (B). The concentration of the DMSO vehicle was identical in all reactions (1% final). The results are the average of three independent experiments and the error bars represent the standard deviation (s.d.).

Mentions: The effect of the compounds on Hog1 activity was evaluated using in vitro kinase assays. For this, we incubated purified in vitro-activated Hog1 together with a biotinylated peptide as a substrate. The initial assays were performed in the presence of 0.1 µM of the compounds 1a–1e, 4a–4e, and SB203580, and the efficacy of these compounds to reduce phosphorylation of the Hog1 substrate was measured (Figure 5A).


Design, synthesis, and characterization of a highly effective Hog1 inhibitor: a powerful tool for analyzing MAP kinase signaling in yeast.

Dinér P, Veide Vilg J, Kjellén J, Migdal I, Andersson T, Gebbia M, Giaever G, Nislow C, Hohmann S, Wysocki R, Tamás MJ, Grøtli M - PLoS ONE (2011)

In vitro Hog1 kinase activity assays.(A) Efficacy of compounds 1a–e,                                4a–e, and SB203580. (B)                                IC50 curves for compounds 4a,                                4b, and SB203580. Kinase assays were                            performed in a kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM                                MgCl2, 2 mM DTT) containing 0.4 µg GST-Hog1, 0.2 mM                            ATP, 0.1 µCi/nmol [32γP]ATP, and 100                            µM peptide substrate, and Hog1 activity was determined as                            described in the Experimental section. Kinase reactions were performed                            in the presence of 0.1 µM inhibitor (A) or with a range of                            inhibitor concentrations (B). The concentration of the DMSO vehicle was                            identical in all reactions (1% final). The results are the                            average of three independent experiments and the error bars represent                            the standard deviation (s.d.).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3104989&req=5

pone-0020012-g005: In vitro Hog1 kinase activity assays.(A) Efficacy of compounds 1a–e, 4a–e, and SB203580. (B) IC50 curves for compounds 4a, 4b, and SB203580. Kinase assays were performed in a kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 2 mM DTT) containing 0.4 µg GST-Hog1, 0.2 mM ATP, 0.1 µCi/nmol [32γP]ATP, and 100 µM peptide substrate, and Hog1 activity was determined as described in the Experimental section. Kinase reactions were performed in the presence of 0.1 µM inhibitor (A) or with a range of inhibitor concentrations (B). The concentration of the DMSO vehicle was identical in all reactions (1% final). The results are the average of three independent experiments and the error bars represent the standard deviation (s.d.).
Mentions: The effect of the compounds on Hog1 activity was evaluated using in vitro kinase assays. For this, we incubated purified in vitro-activated Hog1 together with a biotinylated peptide as a substrate. The initial assays were performed in the presence of 0.1 µM of the compounds 1a–1e, 4a–4e, and SB203580, and the efficacy of these compounds to reduce phosphorylation of the Hog1 substrate was measured (Figure 5A).

Bottom Line: These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo.Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress.Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

View Article: PubMed Central - PubMed

Affiliation: Medicinal Chemistry, Department of Chemistry, University of Gothenburg, Göteborg, Sweden.

ABSTRACT
The Saccharomyces cerevisiae High-Osmolarity Glycerol (HOG) pathway is a conserved mitogen-activated protein kinase (MAPK) signal transduction system that often serves as a model to analyze systems level properties of MAPK signaling. Hog1, the MAPK of the HOG-pathway, can be activated by various environmental cues and it controls transcription, translation, transport, and cell cycle adaptations in response to stress conditions. A powerful means to study signaling in living cells is to use kinase inhibitors; however, no inhibitor targeting wild-type Hog1 exists to date. Herein, we describe the design, synthesis, and biological application of small molecule inhibitors that are cell-permeable, fast-acting, and highly efficient against wild-type Hog1. These compounds are potent inhibitors of Hog1 kinase activity both in vitro and in vivo. Next, we use these novel inhibitors to pinpoint the time of Hog1 action during recovery from G(1) checkpoint arrest, providing further evidence for a specific role of Hog1 in regulating cell cycle resumption during arsenite stress. Hence, we describe a novel tool for chemical genetic analysis of MAPK signaling and provide novel insights into Hog1 action.

Show MeSH
Related in: MedlinePlus