title: A real-time, fluorescence-based assay for Rho-associated protein kinase activity.
abstract: Inhibitors of Rho-associated protein kinase (ROCK) enzymatic activity have been shown to reduce the invasive phenotype observed in metastatic hepatocellular carcinoma (HCC). We describe the design, synthesis, and evaluation of a direct probe for ROCK activity utilizing a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox. The Sox fluorophore undergoes an increase in fluorescence upon phosphorylation of a proximal amino acid via chelation-enhanced fluorescence (CHEF, ex. = 360 nm and em. = 485 nm), allowing for the direct visualization of the rate of phosphate addition to a peptide substrate over time. Our optimal probe design, ROCK-S1, is capable of sensitively reporting ROCK activity with a limit of detection of 10 pM and a high degree of reproducibility (Z'-factor = 0.6 at 100 pM ROCK2). As a proof-of-principle for high-throughput screening (HTS) we demonstrate the ability to rapidly assess the efficacy of a 78 member, small molecule library against ROCK2 using a robotics platform. We identify two previously unreported ROCK2 inhibitor scaffolds, PHA665752 and IKK16, with IC50 values of 3.6 μM and 247 nM respectively. Lastly, we define conditions for selectively monitoring ROCK activity in the presence of potential off-target enzymes (PKCα, PKA, and PAK) with similar substrate specificities.
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States.
authors.forename: Maia I
authors.initials: MI
authors.lastname: Kelly
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States.
authors.forename: Tyler J
authors.initials: TJ
authors.lastname: Bechtel
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States.
authors.forename: D Rajasekhar
authors.initials: DR
authors.lastname: Reddy
authors.affiliation: Department of Chemistry, University of Nebraska - Lincoln, Lincoln, NE 68588, United States.
authors.forename: Erome D
authors.initials: ED
authors.lastname: Hankore
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. Electronic address: cstains2@unl.edu.
authors.forename: Cliff I
authors.initials: CI
authors.lastname: Stains
mesh_terms: D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods; D057075:Enzyme Assays / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D054460:rho-Associated Kinases / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases
keywords: Fluorescence-based biosensor; Inhibition; Kinase activity assay; Rho-associated protein kinase; Small molecule screening