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are consistent with the trans autophosphorylation model because
the cis model predicts selective blockade of autophosphorylation of
the WT receptor.
1
2
3
CONCLUSION
4
5
6
7
8
9
In summary, by covalently targeting a unique reactive feature
in a receptor tyrosine kinase EphB3, we were able to identify in-
hibitors that exhibited high selectivity toward EphB3 not only
among protein kinases but also in the entire proteome. Our rigor-
ous and thorough characterizations verified the exquisite specifici-
ty of these inhibitors, assuring their application in biological stud-
ies. We solved the crystal structures of EphB3 in complex with
covalent inhibitors for the first time. In addition to verifying the
covalent linkage, these covalent complex structures can guide the
design of analogous electrophilic inhibitors for targeting cysteine
in proteins. Beyond generating and characterizing these covalent
EphB3 inhibitors, we employed them to probe drug-target en-
gagement and investigate the mechanism of receptor activation in
an innovative way. Using a clickable probe, we were able to
measure the extent and kinetics of EphB3 engagement by different
types of kinase inhibitors in cells. One specific inhibitor of EphB3
allowed us to reveal that the autophosphorylation of the receptor
in the juxtamembrane segment occurred in trans. These exquisite-
ly specific inhibitors will accelerate pharmacological characteriza-
tion of the roles of EphB3 in various biological processes such as
neural development and tumorigenesis.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.xxxxxxx.
Experimental methods, additional figures and analyses.
AUTHOR INFORMATION
Corresponding Author
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Prof. B. Wang (Case Western University) and F. Sicheri
(University of Toronto) for providing the constructs of EphB3 and
EphA4 respectively. We thank Prof. G. Parkash (University of
Southern California) for providing ephrin-B2 and M. Greenberg
(Harvard University) for anti-phosphoEphB3. We thank Prof. K.
M. Shokat and Ms. B. Levin (University of California, San Fran-
cisco) for help with mass spectrometry data collection. This work
was supported in part by National Science Foundation (CHE-
1455306), American Cancer Society (IRG-58-007-51), and Uni-
versity of Southern California.
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