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PDBsum entry 3gcs
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References listed in PDB file
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Key reference
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Title
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Development of a fluorescent-Tagged kinase assay system for the detection and characterization of allosteric kinase inhibitors.
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Authors
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J.R.Simard,
M.Getlik,
C.Grütter,
V.Pawar,
S.Wulfert,
M.Rabiller,
D.Rauh.
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Ref.
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J Am Chem Soc, 2009,
131,
13286-13296.
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PubMed id
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Abstract
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Kinase disregulation disrupts the intricate network of intracellular signaling
pathways and contributes to the onset of diseases such as cancer. Although
several kinase inhibitors are on the market, inhibitor selectivity and drug
resistance mutations persist as fundamental challenges in the development of
effective long-term treatments. Chemical entities binding to less conserved
allosteric sites would be expected to offer new opportunities for scaffold
development. Because no high-throughput method was previously available, we
developed a fluorescence-based kinase binding assay for identifying and
characterizing ligands which stabilize the inactive kinase conformation. Here,
we present a description of the development and validation of this assay using
the serine/threonine kinase p38alpha. By covalently attaching fluorophores to
the activation loop of the kinase, we were able to detect conformational changes
and measure the K(d), k(on), and k(off) associated with the binding and
dissociation of ligands to the allosteric pocket. We report the SAR of a
synthesized focused library of pyrazolourea derivatives, a scaffold known to
bind with high affinity to the allosteric pocket of p38alpha. Additionally, we
used protein X-ray crystallography together with our assay to examine the
binding and dissociation kinetics to characterize potent quinazoline- and
quinoline-based type II inhibitors, which also utilize this binding pocket in
p38alpha. Last, we identified the b-Raf inhibitor sorafenib as a potent low
nanomolar inhibitor of p38alpha and used protein X-ray crystallography to
confirm a unique binding mode to the inactive kinase conformation.
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