A novel inhibitor of zipper-interacting protein kinase (ZIPK) was used to examine the involvement of ZIPK in the regulation of smooth muscle contraction. Pretreatment of de-endothelialized rat caudal arterial smooth muscle strips with the pyrazolo [3,4-propanamide (HS38) decreased the velocity of contraction (time to reach half-maximal force) induced by the phosphatase inhibitor calyculin A in the presence of Ca 21 without affecting maximal force development. This effect was reversed following washout of HS38 and correlated with a reduction in the rate of phosphorylation of myosin 20-kDa regulatory light chains (LC 20 ) but not of protein kinase C-potentiated inhibitory protein for myosin phosphatase of 17 kDa (CPI-17), prostate apoptosis response-4, or myosin phosphatase-targeting subunit 1 (MYPT1), all of which have been implicated in the regulation of vascular contractility. A structural analog of HS38, with inhibitory activity toward proviral integrations of Moloney (PIM) virus 3 kinase but not ZIPK, had no effect on calyculin A-induced contraction or protein phosphorylations. We conclude that a pool of constitutively active ZIPK is involved in regulation of vascular smooth muscle contraction through direct phosphorylation of LC 20 upon inhibition of myosin light chain phosphatase activity. HS38 also significantly attenuated both phasic and tonic contractile responses elicited by phenylephrine, angiotensin II, endothelin-1, U46619, and K 1 -induced membrane depolarization in the presence of Ca 21 , which correlated with inhibition of phosphorylation of LC 20 , MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Ga 12/13 and Ga q/11 .

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.