Remarkably Stereospecific Utilization of ATP α,ß-Halomethylene Analogues by Protein Kinases.

ATP analogues containing a CXY group in place of the α,ß-bridging oxygen atom are powerful chemical probes for studying ATP-dependent enzymes. A limitation of such probes has been that conventional synthetic methods generate a mixture of diastereomers when the bridging carbon substitution is nonequivalent (X ≠ Y). We report here a novel method based on derivatization of a bisphosphonate precursor with a d-phenylglycine chiral auxiliary that enables preparation of the individual diastereomers of α,ß-CHF-ATP and α,ß-CHCl-ATP, which differ only in the configuration at the CHX carbon. When tested on a dozen divergent protein kinases, these individual diastereomers exhibit remarkable diastereospecificity (up to over 1000-fold) in utilization by the enzymes. This high selectivity can be exploited in an enzymatic approach to obtain the otherwise inaccessible diastereomers of α,ß-CHBr-ATP. The crystal structure of a tyrosine kinase Src bound to α,ß-CHX-ADP establishes the absolute configuration of the CHX carbon and helps clarify the origin of the remarkable diastereospecificity observed. We further synthesized the individual diastereomers of α,ß-CHF-γ-thiol-ATP and demonstrated their utility in labeling a wide spectrum of kinase substrates. The novel ATP substrate analogues afforded by these two complementary strategies should have broad application in the study of the structure and function of ATP-dependent enzymes.

Development of Specific, Irreversible Inhibitors for a Receptor Tyrosine Kinase EphB3.

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 Chemical-Genetic Approach to Generate Selective Covalent Inhibitors of Protein Kinases.

Although a previously developed bump-hole approach has proven powerful in generating specific inhibitors for mapping functions of protein kinases, its application is limited by the intolerance of the large-to-small mutation by certain kinases and the inability to control two kinases separately in the same cells. Herein, we describe the development of an alternative chemical-genetic approach to overcome these limitations. Our approach features the use of an engineered cysteine residue at a particular position as a reactive feature to sensitize a kinase of interest to selective covalent blockade by electrophilic inhibitors and is thus termed the Ele-Cys approach. We successfully applied the Ele-Cys approach to identify selective covalent inhibitors of a receptor tyrosine kinase EphB1 and solved cocrystal structures to determine the mode of covalent binding. Importantly, the Ele-Cys and bump-hole approaches afforded orthogonal inhibition of two distinct kinases in the cell, opening the door to their combined use in the study of multikinase signaling pathways.

Development of Alkyne-Containing Pyrazolopyrimidines To Overcome Drug Resistance of Bcr-Abl Kinase.

Despite the success of imatinib at inhibiting Bcr-Abl and treating chronic myelogenous leukemia (CML), resistance to the therapy occurs over time in patients. In particular, the resistance to imatinib caused by the gatekeeper mutation T315I in Bcr-Abl remains a challenge in the clinic. Inspired by the successful development of ponatinib to curb drug resistance, we hypothesize that the incorporation of an alkyne linker in other heterocyclic scaffolds can also achieve potent inhibition of Bcr-Abl(T315I) by allowing for simultaneous occupancy of both the active site and the allosteric pocket in the Abl kinase domain. Herein, we describe the design, synthesis, and characterization of a series of alkyne-containing pyrazolopyrimidines as Bcr-Abl inhibitors. Our results demonstrate that some alkyne-containing pyrazolopyrimidines potently inhibit not only Abl(T315I) in vitro but also Bcr-Abl(T315I) in cells. These pyrazolopyrimidines can serve as lead compounds for future development of novel targeted therapy to overcome drug resistance of CML.

5'-ß,γ-CHF-ATP diastereomers: synthesis and fluorine-mediated selective binding by c-Src protein kinase.

The first preparation of the individual ß,γ-CHF-ATP stereoisomers 12a and 12b is reported. Configurationally differing solely by the orientation of the C-F fluorine, 12a and 12b have discrete (31)P (202 MHz, pH 10.9, ΔδPα 6 Hz, ΔδPß 4 Hz) and (19)F NMR (470 MHz, pH 9.8, ΔδF 25 Hz) spectral signatures and exhibit a 6-fold difference in IC50 values for c-Src kinase, attributed to a unique interaction of the (S)-fluorine of bound 12b with R388 in the active site.