Abstract
DAPK1 and ZIPK (also called DAPK3) are closely related serine/threonine protein kinases that regulate programmed cell death and phosphorylation of non-muscle and smooth muscle myosin. We have developed a fluorescence linked enzyme chemoproteomic strategy (FLECS) for the rapid identification of inhibitors for any element of the purinome and identified a selective pyrazolo[3,4-d]pyrimidinone (HS38) that inhibits DAPK1 and ZIPK in an ATP-competitive manner at nanomolar concentrations. In cellular studies, HS38 decreased RLC20 phosphorylation. In ex vivo studies, HS38 decreased contractile force generated in mouse aorta, rabbit ileum, and calyculin A stimulated arterial muscle by decreasing RLC20 and MYPT1 phosphorylation. The inhibitor also promoted relaxation in Ca(2+)-sensitized vessels. A close structural analogue (HS43) with 5-fold lower affinity for ZIPK produced no effect on cells or tissues. These findings are consistent with a mechanism of action wherein HS38 specifically targets ZIPK in smooth muscle. The discovery of HS38 provides a lead scaffold for the development of therapeutic agents for smooth muscle related disorders and a chemical means to probe the function of DAPK1 and ZIPK across species.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphate / metabolism
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Animals
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Aorta / cytology
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Aorta / drug effects
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Aorta / enzymology
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Binding, Competitive
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Calcium / metabolism
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Death-Associated Protein Kinases / antagonists & inhibitors*
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Death-Associated Protein Kinases / genetics
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Death-Associated Protein Kinases / metabolism
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Green Fluorescent Proteins / genetics
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Green Fluorescent Proteins / metabolism*
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HEK293 Cells
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High-Throughput Screening Assays
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Humans
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Ileum / cytology
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Ileum / drug effects
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Ileum / enzymology
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Mice
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Muscle Contraction / drug effects
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Muscle, Smooth / cytology
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Muscle, Smooth / drug effects*
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Muscle, Smooth / enzymology
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Myocytes, Smooth Muscle / cytology
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Myocytes, Smooth Muscle / drug effects*
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Myocytes, Smooth Muscle / enzymology
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Myosin-Light-Chain Kinase / antagonists & inhibitors
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Myosin-Light-Chain Kinase / metabolism
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Myosin-Light-Chain Phosphatase
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Phosphorylation
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Primary Cell Culture
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Protein Kinase Inhibitors / chemistry
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Protein Kinase Inhibitors / pharmacology*
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Proteomics
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Pyrazoles / chemistry
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Pyrazoles / pharmacology*
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Pyrimidinones / chemistry
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Pyrimidinones / pharmacology*
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Rabbits
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism*
Substances
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(2-((1-(3-chlorophenyl)-4-oxo-4,5-dihydro-1H-pyrazolo(3,4-d)pyrimidin-6-yl)thio)propanamide
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Protein Kinase Inhibitors
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Pyrazoles
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Pyrimidinones
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Recombinant Fusion Proteins
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Green Fluorescent Proteins
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Adenosine Triphosphate
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DAPK1 protein, human
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Dapk3 protein, mouse
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Death-Associated Protein Kinases
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Myosin-Light-Chain Kinase
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Myosin-Light-Chain Phosphatase
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Ppp1r12a protein, mouse
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Calcium