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Effect of construct design on MAPKAP kinase-2 activity, thermodynamic stability and ligand-binding affinity
Jukka Kervinen1, Hongchang Ma, Shariff Bayoumy
1Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 665 Stockton Drive, Exton, Pennsylvania 19341, USA. jkervine@prdus.jnj.com
Archives of Biochemistry and Biophysics
|April 20, 2006
Summary
MAPK-activated protein kinase-2 (MAPKAPK2) is a drug target for inflammatory diseases. Studies characterized MAPKAPK2 constructs, revealing insights into ligand binding and inhibitor susceptibility for developing selective small-molecule inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- MAPK-activated protein kinase-2 (MAPKAPK2) is crucial for tumor necrosis factor and cytokine synthesis.
- MAPKAPK2 represents a potential therapeutic target for inflammatory diseases.
Purpose of the Study:
- To characterize different MAPKAPK2 protein constructs.
- To investigate the mechanism of ligand binding to MAPKAPK2.
- To provide data for the development of selective small-molecule inhibitors.
Main Methods:
- Production of five MAPKAPK2 protein constructs using baculovirus-infected insect cells.
- Characterization of kinase activity, thermal stability, and ligand-binding affinity.
- Utilized ThermoFluor assay for nucleotide analogue affinity measurements.
Main Results:
- Removal of the C-terminal autoinhibitory peptide affected enzyme stability and activity.
- Phosphorylation of a specific construct increased activity 12-fold.
- Residues 1-40 were essential for high-affinity binding of various ligands; a mutation M138A affected inhibitor susceptibility.
Conclusions:
- These findings elucidate the ligand-binding mechanism of MAPKAPK2.
- The data supports the ongoing search for selective small-molecule inhibitors targeting MAPKAPK2.
- Understanding MAPKAPK2 structure-activity relationships is key for drug development in inflammatory diseases.
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