Abstract
Rho kinase, is the most widely studied downstream effector of the small Rho GTPase RhoA. Two Rho kinase isoforms have been described and are frequently referred to in the literature as ROCK1and ROCK2. The RhoA–Rho kinase pathway has been implicated in the recruitment of cellular infiltrates to disease loci in a number of preclinical animal models of inflammatory disease. In this study, we used biochemical enzyme assays and a cellular target biomarker assay to define PF-4950834 [N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide] as an ATP-competitive, selective Rho kinase inhibitor. We further used PF-4950834 to study the role of Rho kinase activation in lymphocyte and neutrophil migration in addition to the endothelial cell-mediated expression of adhesion molecules and chemokines, which are essential for leukocyte recruitment. The inhibitor blocked stromal cell-derived factor-1α-mediated chemotaxis of T lymphocytes in vitro and the synthesis of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in activated human endothelial cells in vitro. The secretion of chemokines interleukin-8 and monocyte chemoattractant protein-1 was also inhibited in activated endothelial cells. In addition, when dosed orally, the compound potently inhibited neutrophil migration in a carrageenan-induced acute inflammation model. In summary, we have used a pharmacologic approach to link Rho kinase activation to multiple phenotypes that can contribute to leukocyte infiltration. Inhibition of this pathway therefore could be strongly anti-inflammatory and provide therapeutic benefit in chronic inflammatory diseases.
Footnotes
This work was sponsored by Pfizer Inc.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.166033.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- PF-4950834
- N-methyl-3-{[(4-pyridin-4-ylbenzoyl)amino]methyl}benzamide
- AGC
- cAMP-dependent protein kinase/protein kinase G/protein kinase C
- SDF-1α
- stromal cell-derived factor-1α
- CAM
- cell adhesion molecule
- VCAM-1
- vascular cell adhesion molecule-1
- ICAM-1
- intracellular adhesion molecule-1
- IL-8
- interleukin-8
- MCP-1
- monocyte chemoattractant protein-1
- LPA
- lysophosphatidic acid
- MLC
- myosin light chain
- ppMLC
- diphosphorylated MLC
- MYPT-1
- myosin light chain phosphatase-1
- ERM
- ezrin/radixin/moesin
- pERM
- phospho-ERM
- NF-κB
- nuclear factor κB
- HUVEC
- human umbilical vein endothelial cell
- EGM
- endothelial cell growth medium
- GAPDH
- glyceraldehyde phosphate dehydrogenase
- AUC
- area under the curve
- GST
- glutathione S-transferase
- DMSO
- dimethyl sulfoxide
- PBS
- phosphate-buffered saline
- FBS
- fetal bovine serum
- LC
- liquid chromatography
- MS/MS
- tandem mass spectrometry
- mpk
- milligram per kilogram
- PBMC
- peripheral blood mononuclear cell
- PRKG
- cGMP-dependent protein kinase
- PTEN
- phosphatase and tensin homolog
- PK
- pharmacokinetic
- Cmax
- highest plasma concentration measured
- tmax
- time of Cmax.
- Received January 15, 2010.
- Accepted March 11, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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