TY - JOUR
T1 - A real-time, fluorescence-based assay for Rho-associated protein kinase activity
AU - Kelly, Maia I.
AU - Bechtel, Tyler J.
AU - Reddy, D. Rajasekhar
AU - Hankore, Erome D.
AU - Beck, Jon R.
AU - Stains, Cliff I.
N1 - Funding Information:
We acknowledge the Nebraska Center for Mass Spectrometry for assistance with peptide characterization and the HTS facility at the University of Nebraska Medical Center for assistance with proof-of-principle library screening. This work was funded by the Nebraska Research Initiative , the Proposed Center for Integrated Biomolecular Communication , the Fred & Pamela Buffett Cancer Center , and the Department of Chemistry at the University of Nebraska–Lincoln . T. J. Bechtel was supported by an NSF REU grant ( 1156560 ).
Publisher Copyright:
© 2015 Elsevier B.V.
PY - 2015/9/3
Y1 - 2015/9/3
N2 - 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.
AB - 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.
KW - Fluorescence-based biosensor
KW - Inhibition
KW - Kinase activity assay
KW - Rho-associated protein kinase
KW - Small molecule screening
UR - http://www.scopus.com/inward/record.url?scp=84941878278&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84941878278&partnerID=8YFLogxK
U2 - 10.1016/j.aca.2015.07.058
DO - 10.1016/j.aca.2015.07.058
M3 - Article
C2 - 26388388
AN - SCOPUS:84941878278
SN - 0003-2670
VL - 891
SP - 284
EP - 290
JO - Analytica Chimica Acta
JF - Analytica Chimica Acta
ER -