title: The NOX1/4 inhibitor GKT136901 as selective and direct scavenger of peroxynitrite.
abstract: NADPH oxidases (NOX), catalyzing the reduction of molecular oxygen to form the superoxide radical anion (•O₂⁻) and hydrogen peroxide (H₂O₂), are involved in several pathological conditions, such as stroke, diabetes, atherosclerosis, but also in chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, or multiple sclerosis. GKT136901 is a novel NOX-1/4 inhibitor with potential application in the areas of diabetic nephropathy, stroke, or neurodegeneration. In the present study, we investigated additional pharmacological activities of the compound with respect to direct free radical scavenging. GKT136901 did not interact with nitric oxide (•NO), •O₂⁻, or hydroxyl radicals (•OH), but it acted as selective scavenger of peroxynitrite (PON) already in the submicromolar concentration range. Alpha synuclein (ASYN) is a protein involved in the pathogenesis of Parkinson's disease and a known target for PON-dependent tyrosine nitration. Submicromolar concentrations of GKT136901 prevented tyrosine nitration and di-tyrosine-dependent dimer formation of ASYN by PON as indicated by Western blot and mass spectrometric analysis. GKT136901 itself was degraded when exposed to PON. In a human neuronal cell model, GKT136901 prevented both the depletion of reduced intracellular glutathione, and the degeneration of neurites when present during PON treatment of the cells. When GKT136901 was applied after PON treatment, no protective effect was observed, thus excluding an impact of GKT136901 on cellular death/survival pathways. In summary, selective scavenging of PON is an additional pharmacological property of the NOX-1/4 inhibitor GKT136901, and this may add to the efficiency of the drug in several disease models.
authors.forename: S
authors.initials: S
authors.lastname: Schildknecht
authors.forename: A
authors.initials: A
authors.lastname: Weber
authors.forename: H R
authors.initials: HR
authors.lastname: Gerding
authors.forename: R
authors.initials: R
authors.lastname: Pape
authors.forename: M
authors.initials: M
authors.lastname: Robotta
authors.forename: M
authors.initials: M
authors.lastname: Drescher
authors.forename: A
authors.initials: A
authors.lastname: Marquardt
authors.forename: A
authors.initials: A
authors.lastname: Daiber
authors.forename: B
authors.initials: B
authors.lastname: Ferger
authors.affiliation: University of Konstanz; PO Box M657; 78457 Konstanz, Germany. Stefan.Schildknecht@uni-konstanz.de.
authors.forename: M
authors.initials: M
authors.lastname: Leist
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D016166:Free Radical Scavengers / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors*; D019636:Neurodegenerative Diseases / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology*; D011728:Pyridones / Q000494:pharmacology*; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism
chemical_list: C554850:2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo(4,3-c)pyridine-3,6(2H,5H)-dione; D016166:Free Radical Scavengers; D011720:Pyrazoles; D011728:Pyridones; D051844:alpha-Synuclein; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; D000074624:NADPH Oxidase 1; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C402799:NOX1 protein, human; C498910:NOX4 protein, human