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Design, synthesis and characterization of a highly effective inhibitor for analog-sensitive (as) kinases.

Klein M, Morillas M, Vendrell A, Brive L, Gebbia M, Wallace IM, Giaever G, Nislow C, Posas F, Grøtli M - PLoS ONE (2011)

Bottom Line: This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress.This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases.The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

View Article: PubMed Central - PubMed

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

ABSTRACT
Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1(T100G)). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

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Inhibitor docked to Hog1.Compound 6a docked to the homology models of wild-type (red mesh) and T100G mutant (ball-and-stick and yellow surface) Hog1, shown as cross sections from two angles, rotated 90 degrees around the horizontal axis. Compound 6a was designed to occupy the region available only for the T100G mutant (red mesh near phenyl ring) for specificity.
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pone-0020789-g001: Inhibitor docked to Hog1.Compound 6a docked to the homology models of wild-type (red mesh) and T100G mutant (ball-and-stick and yellow surface) Hog1, shown as cross sections from two angles, rotated 90 degrees around the horizontal axis. Compound 6a was designed to occupy the region available only for the T100G mutant (red mesh near phenyl ring) for specificity.

Mentions: Because of its central role in cellular homeostasis and the implication of human homologs in diverse disease states, we selected Hog1 as the target of our mutant kinase-inhibitor pair design. Sequence alignment analyses identified the conserved T100 as a gatekeeper residue in Hog1 [5], [6]. Visual inspection of the binding pocket of an initial homology model of Hog1, using the structure of human p38 in the absence of a ligand (pdb code 1p38) for a template, indicated that a narrow path leads to a buried cavity within the ATP binding domain (Figure 1).


Design, synthesis and characterization of a highly effective inhibitor for analog-sensitive (as) kinases.

Klein M, Morillas M, Vendrell A, Brive L, Gebbia M, Wallace IM, Giaever G, Nislow C, Posas F, Grøtli M - PLoS ONE (2011)

Inhibitor docked to Hog1.Compound 6a docked to the homology models of wild-type (red mesh) and T100G mutant (ball-and-stick and yellow surface) Hog1, shown as cross sections from two angles, rotated 90 degrees around the horizontal axis. Compound 6a was designed to occupy the region available only for the T100G mutant (red mesh near phenyl ring) for specificity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3117834&req=5

pone-0020789-g001: Inhibitor docked to Hog1.Compound 6a docked to the homology models of wild-type (red mesh) and T100G mutant (ball-and-stick and yellow surface) Hog1, shown as cross sections from two angles, rotated 90 degrees around the horizontal axis. Compound 6a was designed to occupy the region available only for the T100G mutant (red mesh near phenyl ring) for specificity.
Mentions: Because of its central role in cellular homeostasis and the implication of human homologs in diverse disease states, we selected Hog1 as the target of our mutant kinase-inhibitor pair design. Sequence alignment analyses identified the conserved T100 as a gatekeeper residue in Hog1 [5], [6]. Visual inspection of the binding pocket of an initial homology model of Hog1, using the structure of human p38 in the absence of a ligand (pdb code 1p38) for a template, indicated that a narrow path leads to a buried cavity within the ATP binding domain (Figure 1).

Bottom Line: This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress.This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases.The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

View Article: PubMed Central - PubMed

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

ABSTRACT
Highly selective, cell-permeable and fast-acting inhibitors of individual kinases are sought-after as tools for studying the cellular function of kinases in real time. A combination of small molecule synthesis and protein mutagenesis, identified a highly potent inhibitor (1-Isopropyl-3-(phenylethynyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine) of a rationally engineered Hog1 serine/threonine kinase (Hog1(T100G)). This inhibitor has been successfully used to study various aspects of Hog1 signaling, including a transient cell cycle arrest and gene expression changes mediated by Hog1 in response to stress. This study also underscores that the general applicability of this approach depends, in part, on the selectivity of the designed the inhibitor with respect to activity versus the engineered and wild type kinases. To explore this specificity in detail, we used a validated chemogenetic assay to assess the effect of this inhibitor on all gene products in yeast in parallel. The results from this screen emphasize the need for caution and for case-by-case assessment when using the Analog-Sensitive Kinase Allele technology to assess the physiological roles of kinases.

Show MeSH