title: Development of Specific, Irreversible Inhibitors for a Receptor Tyrosine Kinase EphB3.
abstract: Erythropoietin-producing human hepatocellular carcinoma (Eph) receptor tyrosine kinases (RTKs) regulate a variety of dynamic cellular events, including cell protrusion, migration, proliferation, and cell-fate determination. Small-molecule inhibitors of Eph kinases are valuable tools for dissecting the physiological and pathological roles of Eph. However, there is a lack of small-molecule inhibitors that are selective for individual Eph isoforms due to the high homology within the family. Herein, we report the development of the first potent and specific inhibitors of a single Eph isoform, EphB3. Through structural bioinformatic analysis, we identified a cysteine in the hinge region of the EphB3 kinase domain, a feature that is not shared with any other human kinases. We synthesized and characterized a series of electrophilic quinazolines to target this unique, reactive feature in EphB3. Some of the electrophilic quinazolines selectively and potently inhibited EphB3 both in vitro and in cells. Cocrystal structures of EphB3 in complex with two quinazolines confirmed the covalent linkage between the protein and the inhibitors. A "clickable" version of an optimized inhibitor was created and employed to verify specific target engagement in the whole proteome and to probe the extent and kinetics of target engagement of existing EphB3 inhibitors. Furthermore, we demonstrate that the autophosphorylation of EphB3 within the juxtamembrane region occurs in trans using a specific inhibitor. These exquisitely specific inhibitors will facilitate the dissection of EphB3's role in various biological processes and disease contribution.
authors.forename: Alvin
authors.initials: A
authors.lastname: Kung
authors.forename: Ying-Chu
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca , Building 310, Cambridge Science Park, Milton Road, Cambridge CB4 0WG, United Kingdom.
authors.forename: Marianne
authors.initials: M
authors.lastname: Schimpl
authors.forename: Feng
authors.initials: F
authors.lastname: Ni
authors.forename: Jianfa
authors.initials: J
authors.lastname: Zhu
authors.forename: Maurice
authors.initials: M
authors.lastname: Turner
authors.affiliation: Proteomic Resource Center, The Rockefeller University , New York, New York 10065, United States.
authors.forename: Henrik
authors.initials: H
authors.lastname: Molina
authors.affiliation: Discovery Sciences, Innovative Medicines and Early Development Biotech Unit, AstraZeneca , Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom.
authors.forename: Ross
authors.initials: R
authors.lastname: Overman
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D015195:Drug Design; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D036223:Receptor, EphB3 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D036223:Receptor, EphB3