Abstract
Minocycline prevents oxidative protein modifications and damage in disease models associated with inflammatory glial activation and oxidative stress. Although the drug has been assumed to act by preventing the up-regulation of proinflammatory enzymes, we probed here its direct chemical interaction with reactive oxygen species. The antibiotic did not react with superoxide or (•)NO radicals, but peroxynitrite (PON) was scavenged in the range of ∼1 μm minocycline and below. The interaction of pharmacologically relevant minocycline concentrations with PON was corroborated in several assay systems and significantly exceeded the efficacy of other antibiotics. Minocycline was degraded during the reaction with PON, and the resultant products lacked antioxidant properties. The antioxidant activity of minocycline extended to cellular systems, because it prevented neuronal mitochondrial DNA damage and glutathione depletion. Maintenance of neuronal viability under PON stress was shown to be solely dependent on direct chemical scavenging by minocycline. We chose α-synuclein (ASYN), known from Parkinsonian pathology as a biologically relevant target in chemical and cellular nitration reactions. Submicromolar concentrations of minocycline prevented tyrosine nitration of ASYN by PON. Mass spectrometric analysis revealed that minocycline impeded nitrations more effectively than methionine oxidations and dimerizations of ASYN, which are secondary reactions under PON stress. Thus, PON scavenging at low concentrations is a novel feature of minocycline and may help to explain its pharmacological activity.
Highlights
Neuroprotection [4, 5]
Because neurodegenerative diseases are always accompanied by inflammatory conditions [29], the potentially combined direct and indirect actions of minocycline would explain the good activity seen in animal models of disease
Reactive oxygen species generated in many cells under such conditions comprise H2O2, the hydroxyl radical (1⁄7OH), nitric oxide (1⁄7NO), superox
Summary
Materials—Sin-1 (3-morpholinosydnonimine) (peroxynitrite generator), Spermine-NONOate (nitric oxide donor), and authentic peroxynitrite (PON) were purchased from Cayman Chemicals (Ann Arbor, MI). In cell-free experiments, PON (in NaOH) and the corresponding volume of equimolar HCl were added carefully as separate droplets in the inner ring of a reaction tube, closed, and vortexed instantaneously and rigorously. The radical generating systems were incubated with the test compounds for 5 min at 37 °C before the radical detection dyes DHR 123 or L-012 were added and incubated for 15 min. Both dyes were chosen, because they are relatively selective for steady state PON levels in the submicromolar range. The secondary antibody (antimouse-IgG, Alexa 488, Molecular Probes; 1:1000) in 1% BSA/ PBS was added for 1 h, the nuclei were stained by Hoechst dye H-33342 (1 g/ml) for 20 min. The means were considered as statistically significant at p Ͻ 0.05
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