title: Structure-guided development of affinity probes for tyrosine kinases using chemical genetics.
abstract: 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.
authors.affiliation: Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA.
authors.forename: Jimmy A
authors.initials: JA
authors.lastname: Blair
authors.forename: Daniel
authors.initials: D
authors.lastname: Rauh
authors.forename: Charles
authors.initials: C
authors.lastname: Kung
authors.forename: Cai-Hong
authors.initials: CH
authors.lastname: Yun
authors.forename: Qi-Wen
authors.initials: QW
authors.lastname: Fan
authors.forename: Haridas
authors.initials: H
authors.lastname: Rode
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Eck
authors.forename: William A
authors.initials: WA
authors.lastname: Weiss
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D000081247:CSK Tyrosine-Protein Kinase; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D041681:NIH 3T3 Cells; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics*; D011799:Quinazolines / Q000138:chemical synthesis / Q000737:chemistry; D015398:Signal Transduction; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D019061:src-Family Kinases
chemical_list: D000345:Affinity Labels; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases