title: A Small Molecule Pyrazolo[3,4-d]Pyrimidinone Inhibitor of Zipper-Interacting Protein Kinase Suppresses Calcium Sensitization of Vascular Smooth Muscle.
abstract: 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-d]pyrimidinone inhibitor 2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo [3,4-d]-pyrimidin-6-yl)thio)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(2+) 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 (LC20) 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 LC20 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(+)-induced membrane depolarization in the presence of Ca(2+), which correlated with inhibition of phosphorylation of LC20, MYPT1, and CPI-17. These effects of HS38 suggest that ZIPK also lies downstream from G protein-coupled receptors that signal through both Gα12/13 and Gαq/11.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.) jmacdo@ucalgary.ca.
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
authors.forename: Cindy
authors.initials: C
authors.lastname: Sutherland
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
authors.forename: David A
authors.initials: DA
authors.lastname: Carlson
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
authors.forename: Sabreena
authors.initials: S
authors.lastname: Bhaidani
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
authors.forename: Abdulhameed
authors.initials: A
authors.lastname: Al-Ghabkari
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
authors.forename: Karl
authors.initials: K
authors.lastname: Swärd
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
authors.forename: Timothy A J
authors.initials: TA
authors.lastname: Haystead
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Calgary, Cumming School of Medicine, Calgary, Alberta, Canada (J.A.M., C.S., S.B., A.A.-G., M.P.W.); Department of Experimental Medical Science, Lund University, Lund, Sweden (K.S.); and Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina (D.A.C., T.A.J.H.).
authors.forename: Michael P
authors.initials: MP
authors.lastname: Walsh
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D009924:Organ Culture Techniques; D011720:Pyrazoles / Q000737:chemistry / Q000494:pharmacology*; D011743:Pyrimidines / Q000737:chemistry / Q000494:pharmacology*; D011744:Pyrimidinones / Q000737:chemistry / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: C000590333:(2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo(3,4-d)pyrimidin-6-yl)thio)propanamide; D011720:Pyrazoles; D011743:Pyrimidines; D011744:Pyrimidinones; C014175:pyrazolo(3,4-d)pyrimidine; D020930:MAP Kinase Kinase Kinases; C090757:mitogen-activated protein kinase kinase kinase 12; D002118:Calcium