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.

A Scaffold‐Hopping Strategy toward the Identification of Inhibitors of Cyclin G Associated Kinase

“…Condensation of methyl 2‐amino‐4‐bromobenzoate 70 with triethyl orthoformate as a one‐carbon fragment and ammonium acetate as nitrogen source allowed the isolation of 7‐bromoquinazolin‐4(3 H )‐one 71 (Scheme ) . The tautomeric hydroxy group of the lactam moiety was converted into a chlorine by treatment with phosphorus oxychloride, yielding a 4‐chloro‐quinazoline analogue 72 . A nucleophilic aromatic substitution with morpholine yielded the 4‐morpholino‐quinazoline 73 .…”

“…[33] The tautomeric hydroxy group of the lactam moiety was converted into ac hlorine by treatment with phosphorus oxychloride, yielding a4chloro-quinazolinea nalogue 72. [34] An ucleophilic aromatic substitution with morpholine yielded the 4-morpholino-quinazoline 73.C oupling of 73 with 3,4-dimethoxyphenylboronic acid using the aforementioned conditions for the Suzuki reaction furnished quinazoline 74 in good yield.…”

Therapeutic potential of targeting the Eph/ephrin signaling complex

“…Although few have entered clinical trials, the relative infancy of the field and the enormous potential of various ‘druggable’ domains, should guarantee the continuation of these efforts. Kinase inhibitors, specific for the Eph receptors, as well as siRNA molecules that downregulate the expression levels of the Eph’s, have been described elsewhere (Amero et al, 2016; Biao-xue et al, 2011; Boyd et al, 2014; Dong et al, 2015; Duan et al, 2017; Heinzlmeir et al, 2017; Kung et al, 2016; Unzue et al, 2016; Zang et al, 2016) and will not be discussed further.…”

Das Cysteinom der Proteinkinasen als Zielstruktur in der Arzneistoffentwicklung

“…Die Autoren entwickelten außerdem eine “klickbare” Version des Inhibitors, mit dem die zelluläre Selektivität für EphB3 gezeigt werden konnte. Die kovalente Bindung an das erwähnte Cystein wurde röntgenkristallographisch nachgewiesen (Abbildung ) . Auf eine ähnliche Art wurde hohe Selektivität für die Lipidkinase PI3Kα erreicht, indem das F4‐Cystein adressiert wurde (Abbildung ) …”