Detection of myosin light chain phosphorylation--a cell-based assay for screening Rho-kinase inhibitors.
Here, we describe the first example of a cell-based myosin light chain phosphorylation assay in 96-well format that allows for the rapid screening of novel Rho-kinase inhibitors. We obtained IC(50) values for the prototypic Rho-kinase inhibitors Y-27632 (1.2+/-0.05microM) and Fasudil (3.7+/-1.2microM) that were similar to those previously published utilizing electrophoresis-based methodologies. H-1152P, a Fasudil analog showed an IC(50) value of 77+/-30nM. Data derived from a set of 21 novel Rho-kinase inhibitors correlate with those generated by a well-established cell-based phenotypic Rho-kinase inhibition assay (R(2)=0.744). These results show that imaging technology measuring changes in myosin light chain phosphorylation can be used to rapidly generate quantitative IC(50) values and to screen a larger set of small molecule Rho-kinase inhibitors and suggests that this approach can be broadly applied to other cell lines and signaling pathways.
Schröter
Thomas
T
Translational Research Institute, The Scripps Research Institute, Scripps Florida, 5353 Parkside Drive, RF-1 Jupiter, FL 33458, USA. tschroet@scripps.edu
Griffin
Evelyn
E
Weiser
Amiee
A
Feng
Yangbo
Y
LoGrasso
Philip
P
D000577:Amides / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D018994:Myosin Light Chains / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification* / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors*
D000577:Amides; D018994:Myosin Light Chains; D010750:Phosphoproteins; D047428:Protein Kinase Inhibitors; D011725:Pyridines; C108830:Y 27632; D054460:rho-Associated Kinases