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.
Highlights
Smooth muscle plays an important role in the regulation of vascular tone and many other biological functions
There is potential value for the DI compound to study the biological actions of zipper-interacting protein kinase (ZIPK), our results suggest the overlapping functions of ROCKII and ZIPK in VSM cells (VSMCs) contractile mechanisms preclude the use of DI in this context
Two additional DI analogues with ZIPK and DAPK1 inhibitory potential that were previously disclosed by Okamoto and colleagues[41,42], namely (4Z)-2-ethylidene-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5-one (DI-2) and (4Z)-2-(3-methylphenyl)-4-(pyridin-3-ylmethylidene)-1,3-oxazol-5one (DI-3), inhibited the activity of ROCKII(5-554) (Fig. 1B,C, respectively)
Summary
The DI compound is a competitive inhibitor of ROCKII. The (4Z)-2-(4-chloro-3-nitrophenyl)-4-. The potential influence of ZIPK on the regulation of cofilin was interrogated because the protein kinase could be inhibited by DI41,42 Another ATP-competitive ZIPK inhibitor (HS38; IC50 < 200 nM) does not target ROCKII in vitro when examined by 33P-ATP filter binding assay[43] and does not inhibit LC20 phosphorylation by ROCKII when assessed with 32P-phosphoryl transfer assays[29]. The application of a similar concentration (50 μM) of the HS38 inhibitor revealed no significant changes in the phosphorylation levels of cofilin (Fig. 7C) These results suggest that the changes in cofilin phosphorylation upon administration of DI to CASMCs were exclusively associated with ROCKII and not ZIPK. Similar effects on F-actin architecture and pS3-cofilin immunostaining were observed following treatment of CASMCs with known ROCK inhibitors: GSK269962A (Fig. 8C,G) and H1152 (Fig. 8D,H)
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