TY - JOUR
T1 - Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK–Paxillin Interaction
AU - Marlowe, Timothy
AU - Alvarado, Carlos
AU - Rivera, Andrew
AU - Lenzo, Felicia
AU - Nott, Rohini
AU - Bondugji, Dena
AU - Montoya, Justin
AU - Hurley, Alana
AU - Kaplan, Matt
AU - Capaldi, Andrew
AU - Cance, William
N1 - Funding Information:
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: National Cancer Institute (Grant No. R01 CA065910 to William Cance and Timothy Marlowe).
Funding Information:
We would like to thank Dr. David Azorsa for use of the NCI Diversity Set V for pilot screening studies. The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: National Cancer Institute (Grant No. R01 CA065910 to William Cance and Timothy Marlowe).
Publisher Copyright:
© 2019 Society for Laboratory Automation and Screening.
PY - 2020/1/1
Y1 - 2020/1/1
N2 - Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.
AB - Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.
KW - FAT domain
KW - drug discovery
KW - focal adhesion kinase
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U2 - 10.1177/2472555219874313
DO - 10.1177/2472555219874313
M3 - Article
C2 - 31513463
AN - SCOPUS:85072300769
VL - 25
SP - 21
EP - 32
JO - SLAS Discovery
JF - SLAS Discovery
SN - 2472-5552
IS - 1
ER -