3SVV

Crystal Structure of T338C c-Src covalently bound to vinylsulfonamide-pyrazolopyrimidine 9

Summary for 3SVV

• Entry DOI: 10.2210/pdb3svv/pdb
• Descriptor: Proto-oncogene tyrosine-protein kinase Src, N-(3-{[4-amino-1-(propan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-yl]methyl}phenyl)ethanesulfonamide (3 entities in total)
• Functional Keywords: covalently bound ligand, cysteine gatekeeper, src, transferase-transferase inhibitor complex, transferase/transferase inhibitor
• Biological source: Gallus gallus (bantam,chickens)
• Cellular location: Cell membrane (By similarity): P00523
• Total number of polymer chains: 2
• Total formula weight: 66206.29

Authors

Garske, A.L.,Peters, U.,Cortesi, A.,Perez, J.,Shokat, K.M. (deposition date: 2011-07-12, release date: 2011-08-17, Last modification date: 2024-03-13)

Primary citation

Garske, A.L.,Peters, U.,Cortesi, A.T.,Perez, J.L.,Shokat, K.M.
Chemical genetic strategy for targeting protein kinases based on covalent complementarity.
Proc.Natl.Acad.Sci.USA, 108:15046-15052, 2011

PubMed Abstract: 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.

PubMed: 21852571
DOI: 10.1073/pnas.1111239108

Experimental method

X-RAY DIFFRACTION (2.204 Å)