title: A novel inhibitory effect of oxazol-5-one compounds on ROCKII signaling in human coronary artery vascular smooth muscle cells.
abstract: The selectivity of (4Z)-2-(4-chloro-3-nitrophenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI) for zipper-interacting protein kinase (ZIPK) was previously described by in silico computational modeling, screening a large panel of kinases, and determining the inhibition efficacy. Our assessment of DI revealed another target, the Rho-associated coiled-coil-containing protein kinase 2 (ROCKII). In vitro studies showed DI to be a competitive inhibitor of ROCKII (Ki, 132 nM with respect to ATP). This finding was supported by in silico molecular surface docking of DI with the ROCKII ATP-binding pocket. Time course analysis of myosin regulatory light chain (LC20) phosphorylation catalyzed by ROCKII in vitro revealed a significant decrease upon treatment with DI. ROCKII signaling was investigated in situ in human coronary artery vascular smooth muscle cells (CASMCs). ROCKII down-regulation using siRNA revealed several potential substrates involved in smooth muscle contraction (e.g., LC20, Par-4, MYPT1) and actin cytoskeletal dynamics (cofilin). The application of DI to CASMCs attenuated LC20, Par-4, LIMK, and cofilin phosphorylations. Notably, cofilin phosphorylation was not significantly decreased with a novel ZIPK selective inhibitor (HS-38). In addition, CASMCs treated with DI underwent cytoskeletal changes that were associated with diminution of cofilin phosphorylation. We conclude that DI is not selective for ZIPK and is a potent inhibitor of ROCKII.
authors: Department of Biochemistry &Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada.
authors: Abdulhameed
authors: A
authors: Al-Ghabkari
authors: Department of Biochemistry &Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada.
authors: Jing-Ti
authors: JT
authors: Deng
authors: Department of Integrative Oncology, BC Cancer Research Centre, 675 West 10th Ave, Vancouver, BC, V5Z 1L3, Canada.
authors: Paul C
authors: PC
authors: McDonald
authors: Department of Integrative Oncology, BC Cancer Research Centre, 675 West 10th Ave, Vancouver, BC, V5Z 1L3, Canada.
authors: Shoukat
authors: S
authors: Dedhar
authors: Department of Biochemistry &Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada.
authors: Mana
authors: M
authors: Alshehri
authors: Department of Biochemistry &Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada.
authors: Michael P
authors: MP
authors: Walsh
authors: Department of Biochemistry &Molecular Biology, University of Calgary, 3280 Hospital Drive NW, Calgary, AB, T2N 4Z6, Canada.
authors: Justin A
authors: JA
authors: MacDonald
mesh_terms: D003331:Coronary Vessels / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D062105:Molecular Docking Simulation; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000201:enzymology*; D010080:Oxazoles / Q000737:chemistry / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects*; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
chemical_list: D010080:Oxazoles; D047428:Protein Kinase Inhibitors; C515852:ROCK2 protein, human; D054460:rho-Associated Kinases