title: Design and evaluation of a real-time activity probe for focal adhesion kinase.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States.
authors.forename: Jon R
authors.initials: JR
authors.lastname: Beck
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States.
authors.forename: Xinqi
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States.
authors.forename: Garrett R
authors.initials: GR
authors.lastname: Casey
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States. Electronic address: cstains2@unl.edu.
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
mesh_terms: D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006801:Humans; D057230:Limit of Detection; D015335:Molecular Probes / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D013997:Time Factors
chemical_list: D015335:Molecular Probes; D047428:Protein Kinase Inhibitors; D051416:Focal Adhesion Protein-Tyrosine Kinases
keywords: Fluorescence-based biosensor; Focal adhesion kinase; Inhibition; Kinase activity assay; Small molecule screening