Date of this Version
Hepatocellular carcinoma (HCC) is an extremely aggressive form of liver cancer with a low survival rate due to high recurrence. Increases in Rho-associated Protein Kinase (ROCK) activity are correlated with a more aggressive, metastatic phenotype associated with advanced HCC. Inhibitors for ROCK have shown potential for the reduction of this metastatic phenotype of HCC. We outline the design and optimization of a direct activity sensor for ROCK that utilizes a phosphorylation-sensitive sulfonamido-oxine fluorophore, termed Sox, and is capable of reporting on the inhibition of ROCK. This CSox-based activity probe utilizes chelation-enhanced fluorescence (ex. = 360 nm and em. = 485 nm) between the proximal phosphorylated residue, Mg2+ and the Sox fluorophore. This allows for the direct and continuous monitoring of phosphorylation of the peptide-based probe over time. The sensitivity of the optimal CSox-based probe, ROCK-S1, was detected to be 10 pM of recombinant enzyme. Using this probe we demonstrate the ability to directly and rapidly assess a pilot small molecule library for inhibitors of ROCK2, using a robotics platform. In a step towards applying our probe in complex biological systems, we identify the optimal conditions for monitoring ROCK2 while inhibiting off-target enzymes (PKCα, PKA, and PAK). Our work provides a sensitive assay platform for ROCK activity that is compatible to HTS and could potentially be used to interrogate ROCK activity in heterogeneous biological samples.
Advisor: Cliff I. Stains