Detection of myosin light chain phosphorylation--a cell-based assay for screening Rho-kinase inhibitors |
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Authors: | Schröter Thomas Griffin Evelyn Weiser Amiee Feng Yangbo LoGrasso Philip |
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Institution: | Translational Research Institute, The Scripps Research Institute, Scripps Florida, 5353 Parkside Drive, RF-1 Jupiter, FL, 33458, USA |
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Abstract: | 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 IC50 values for the prototypic Rho-kinase inhibitors Y-27632 (1.2 ± 0.05 μM) and Fasudil (3.7 ± 1.2 μM) that were similar to those previously published utilizing electrophoresis-based methodologies. H-1152P, a Fasudil analog showed an IC50 value of 77 ± 30 nM. 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 (R2 = 0.744). These results show that imaging technology measuring changes in myosin light chain phosphorylation can be used to rapidly generate quantitative IC50 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. |
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Keywords: | Myosin light chain MLC Rho-kinase ROCK Cell-based assay Thr18 Ser19 Phosphorylation |
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