Limits...
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.

Show MeSH

Related in: MedlinePlus

Uptake of inhibitors by yeast cells.(A) A lawn of wild-type yeast cells (BY4741 strain) was spread on solid                            medium in the absence (control) or presence of osmotic stress (1.5 M                            sorbitol), and filter discs containing various concentrations of                                4a, 4b, and SB203580 were                            placed on top of the lawn. Inhibition of Hog1 activity could be                            visualized by the formation of a halo of non-proliferating cells around                            the filter discs in the presence of osmotic stress (1.5 M sorbitol). No                            such halo was visible on control plates in the absence of osmotic                            stress. Plates were incubated for 48 hours at 30°C. (B)                                4a improves growth of PBS2                            overexpressing cells. Wild-type and hog1Δ cells                            (BY4743 strain) were transformed with an empty plasmid or plasmid                            overexpressing PBS2. Cells were grown in a                            microcultivation system in the absence or presence of inhibitor as                            indicated.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3104989&req=5

pone-0020012-g006: Uptake of inhibitors by yeast cells.(A) A lawn of wild-type yeast cells (BY4741 strain) was spread on solid medium in the absence (control) or presence of osmotic stress (1.5 M sorbitol), and filter discs containing various concentrations of 4a, 4b, and SB203580 were placed on top of the lawn. Inhibition of Hog1 activity could be visualized by the formation of a halo of non-proliferating cells around the filter discs in the presence of osmotic stress (1.5 M sorbitol). No such halo was visible on control plates in the absence of osmotic stress. Plates were incubated for 48 hours at 30°C. (B) 4a improves growth of PBS2 overexpressing cells. Wild-type and hog1Δ cells (BY4743 strain) were transformed with an empty plasmid or plasmid overexpressing PBS2. Cells were grown in a microcultivation system in the absence or presence of inhibitor as indicated.

Mentions: To be useful in vivo, the inhibitors need to enter yeast cells. To test this, a lawn of yeast cells was spread on solid medium and filter discs containing various concentrations of compounds 4a, 4b, and SB203580 were placed on top of the lawn (Figure 6A). Survival of yeast cells in the presence of osmotic stress requires Hog1 activity; hence, inhibition of Hog1 can be visualized by the formation of a halo of non-proliferating cells around the filter discs in the presence of osmotic stress. Thus, the size of the halo is a measure of Hog1 inhibition in this assay.


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)

Uptake of inhibitors by yeast cells.(A) A lawn of wild-type yeast cells (BY4741 strain) was spread on solid                            medium in the absence (control) or presence of osmotic stress (1.5 M                            sorbitol), and filter discs containing various concentrations of                                4a, 4b, and SB203580 were                            placed on top of the lawn. Inhibition of Hog1 activity could be                            visualized by the formation of a halo of non-proliferating cells around                            the filter discs in the presence of osmotic stress (1.5 M sorbitol). No                            such halo was visible on control plates in the absence of osmotic                            stress. Plates were incubated for 48 hours at 30°C. (B)                                4a improves growth of PBS2                            overexpressing cells. Wild-type and hog1Δ cells                            (BY4743 strain) were transformed with an empty plasmid or plasmid                            overexpressing PBS2. Cells were grown in a                            microcultivation system in the absence or presence of inhibitor as                            indicated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3104989&req=5

pone-0020012-g006: Uptake of inhibitors by yeast cells.(A) A lawn of wild-type yeast cells (BY4741 strain) was spread on solid medium in the absence (control) or presence of osmotic stress (1.5 M sorbitol), and filter discs containing various concentrations of 4a, 4b, and SB203580 were placed on top of the lawn. Inhibition of Hog1 activity could be visualized by the formation of a halo of non-proliferating cells around the filter discs in the presence of osmotic stress (1.5 M sorbitol). No such halo was visible on control plates in the absence of osmotic stress. Plates were incubated for 48 hours at 30°C. (B) 4a improves growth of PBS2 overexpressing cells. Wild-type and hog1Δ cells (BY4743 strain) were transformed with an empty plasmid or plasmid overexpressing PBS2. Cells were grown in a microcultivation system in the absence or presence of inhibitor as indicated.
Mentions: To be useful in vivo, the inhibitors need to enter yeast cells. To test this, a lawn of yeast cells was spread on solid medium and filter discs containing various concentrations of compounds 4a, 4b, and SB203580 were placed on top of the lawn (Figure 6A). Survival of yeast cells in the presence of osmotic stress requires Hog1 activity; hence, inhibition of Hog1 can be visualized by the formation of a halo of non-proliferating cells around the filter discs in the presence of osmotic stress. Thus, the size of the halo is a measure of Hog1 inhibition in this assay.

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