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PDBsum entry 2j5f
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References listed in PDB file
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Key reference
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Title
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Structure-Guided development of affinity probes for tyrosine kinases using chemical genetics.
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Authors
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J.A.Blair,
D.Rauh,
C.Kung,
C.H.Yun,
Q.W.Fan,
H.Rode,
C.Zhang,
M.J.Eck,
W.A.Weiss,
K.M.Shokat.
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Ref.
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Nat Chem Biol, 2007,
3,
229-238.
[DOI no: ]
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PubMed id
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Abstract
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As key components in nearly every signal transduction pathway, protein kinases
are attractive targets for the regulation of cellular signaling by
small-molecule inhibitors. We report the structure-guided development of
6-acrylamido-4-anilinoquinazoline irreversible kinase inhibitors that potently
and selectively target rationally designed kinases bearing two selectivity
elements that are not found together in any wild-type kinase: an
electrophile-targeted cysteine residue and a glycine gatekeeper residue.
Cocrystal structures of two irreversible quinazoline inhibitors bound to either
epidermal growth factor receptor (EGFR) or engineered c-Src show covalent
inhibitor binding to the targeted cysteine (Cys797 in EGFR and Cys345 in
engineered c-Src). To accommodate the new covalent bond, the quinazoline core
adopts positions that are different from those seen in kinase structures with
reversible quinazoline inhibitors. Based on these structures, we developed a
fluorescent 6-acrylamido-4-anilinoquinazoline affinity probe to report the
fraction of kinase necessary for cellular signaling, and we used these reagents
to quantitate the relationship between EGFR stimulation by EGF and its
downstream outputs-Akt, Erk1 and Erk2.
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Figure 3.
ESI-oa-TOF of control and drug-treated c-Src kinases are
shown.
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Figure 4.
The experimental electron densities of EGFR and c-Src-cys at
2.95 Å and 2.48 Å resolution, respectively, are
shown (2F[o] – F[c] map contoured at 1  ).
(a) Optimized EGFR inhibitor 2 (PD 168393)^13 (green ball and
sticks) in complex with the EGFR kinase domain shows clear
electron density between the targeted Cys797 and the acrylamide
Michael acceptor on 2. The inhibitor makes a direct hydrogen
bond between its quinazoline N1 and the main chain amide of
Met793, which is a common recognition motif among reversible
anilinoquinazolines and several protein kinase domains^16, ^20,
^21, ^22, ^23, ^24. We find the cocrystal complex in an active
conformation of EGFR: a conserved salt bridge found only in
active EGFR conformations between the catalytic Lys745 and helix
C Glu762 remains intact. The m-bromine group sits adjacent
(within 3.4 Å) to the gatekeeper residue, Thr790. (b)
Notably, structures of 2 in complex with c-Src-cys show an
inhibitor binding mode distinct from any reported for
anilinoquinazolines with a protein kinase. In molecule B of the
c-Src-cys–2 complex, 2 (green ball and sticks) forms a
water-mediated (W1) hydrogen bond via its N1 of the quinazoline
core to the backbone amide of Met341.
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The above figures are
reprinted
by permission from Macmillan Publishers Ltd:
Nat Chem Biol
(2007,
3,
229-238)
copyright 2007.
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