TY - JOUR
T1 - Design and evaluation of a real-time activity probe for focal adhesion kinase
AU - Beck, Jon R.
AU - Zhou, Xinqi
AU - Casey, Garrett R.
AU - Stains, Cliff I.
N1 - Funding Information:
The authors gratefully acknowledge funding from the Elsa U. Pardee Foundation , the Nebraska Research Initiative , the UNL CIBC Research Cluster Development Grant, and the Department of Chemistry at the University of Nebraska – Lincoln . We thank D. Rajasekhar Reddy for helpful conversations. In addition, we acknowledge the Nebraska Center for Mass Spectrometry for assistance with peptide characterization.
Publisher Copyright:
© 2015 Elsevier B.V.
PY - 2015/10/15
Y1 - 2015/10/15
N2 - Focal adhesion kinase (FAK) has been identified as a potential therapeutic target for the treatment of metastatic cancers. Herein we describe the design, synthesis and optimization of a direct activity sensor for FAK and its application to screening FAK inhibitors. We find that the position of the sensing moiety, a phosphorylation-sensitive sulfonamido-oxine fluorophore, can dramatically influence the performance of peptide sensors for FAK. Real-time fluorescence activity assays using an optimized sensor construct, termed FAKtide-S2, are highly reproducible (Z' = 0.91) and are capable of detecting as little as 1 nM recombinant FAK. Utilizing this robust assay format, we define conditions for the screening of FAK inhibitors and demonstrate the utility of this platform using a set of well-characterized small molecule kinase inhibitors. Additionally, we provide the selectivity profile of FAKtide-S2 among a panel of closely related enzymes, identifying conditions for selectively monitoring FAK activity in the presence of off-target enzymes. In the long term, the chemosensor platform described in this work can be used to identify novel FAK inhibitor scaffolds and potentially assess the efficacy of FAK inhibitors in disease models.
AB - Focal adhesion kinase (FAK) has been identified as a potential therapeutic target for the treatment of metastatic cancers. Herein we describe the design, synthesis and optimization of a direct activity sensor for FAK and its application to screening FAK inhibitors. We find that the position of the sensing moiety, a phosphorylation-sensitive sulfonamido-oxine fluorophore, can dramatically influence the performance of peptide sensors for FAK. Real-time fluorescence activity assays using an optimized sensor construct, termed FAKtide-S2, are highly reproducible (Z' = 0.91) and are capable of detecting as little as 1 nM recombinant FAK. Utilizing this robust assay format, we define conditions for the screening of FAK inhibitors and demonstrate the utility of this platform using a set of well-characterized small molecule kinase inhibitors. Additionally, we provide the selectivity profile of FAKtide-S2 among a panel of closely related enzymes, identifying conditions for selectively monitoring FAK activity in the presence of off-target enzymes. In the long term, the chemosensor platform described in this work can be used to identify novel FAK inhibitor scaffolds and potentially assess the efficacy of FAK inhibitors in disease models.
KW - Fluorescence-based biosensor
KW - Focal adhesion kinase
KW - Inhibition
KW - Kinase activity assay
KW - Small molecule screening
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U2 - 10.1016/j.aca.2015.09.025
DO - 10.1016/j.aca.2015.09.025
M3 - Article
C2 - 26515006
AN - SCOPUS:84944894208
SN - 0003-2670
VL - 897
SP - 62
EP - 68
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
ER -