Proceedings of the National Academy of Sciences - PNAS, 2011-09, Vol.108 (37), p.15046-15052
2011
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- Autor(en) / Beteiligte
- Titel
- Chemical genetic strategy for targeting protein kinases based on covalent complementarity
- Ist Teil von
- Proceedings of the National Academy of Sciences - PNAS, 2011-09, Vol.108 (37), p.15046-15052
- Ort / Verlag
- United States: National Academy of Sciences
- Erscheinungsjahr
- 2011
- Quelle
- MEDLINE
- Beschreibungen/Notizen
- The conserved nature of the ATP-binding site of the > 500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few off-targets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 0027-8424eISSN: 1091-6490DOI: 10.1073/pnas.1111239108Titel-ID: cdi_pnas_primary_108_37_15046
- Format
- –
- Schlagworte
- Adenosine triphosphatase, adenosine triphosphate, Alanine, Amino acids, Animals, ATP, Binding sites, Biological Sciences, Cell lines, Chemical compounds, Chemicals, Complementarity, Crystallography, X-Ray, Cysteine, Cysteine - metabolism, Electrical engineering, Enzymatic activity, enzyme activity, Genetic mutation, Genetic Techniques, Genetics, Glycine, Human genetics, Humans, Hydrophobicity, Inhibitory Concentration 50, Kinases, Kinetics, MAP kinase, Medical genetics, Mice, mitogen-activated protein kinase, Models, Molecular, Molecules, Mutation, Mutation - genetics, NIH 3T3 Cells, Physical Sciences, Protein engineering, Protein Kinase Inhibitors - chemistry, Protein Kinase Inhibitors - pharmacology, Protein Kinases - metabolism, Protein-tyrosine kinase, Proteins, pyrazolopyrimidines, Src protein, src-Family Kinases - antagonists & inhibitors, src-Family Kinases - metabolism, tyrosine