N10 -carbonyl-substituted phenothiazines inhibiting lipid peroxidation and associated nitric oxide consumption powerfully protect brain tissue against oxidative stress.

During some investigations into the mechanism of nitric oxide consumption by brain preparations, several potent inhibitors of this process were identified. Subsequent tests revealed the compounds act by inhibiting lipid peroxidation, a trigger for a form of regulated cell death known as ferroptosis. A quantitative structure-activity study together with XED (eXtended Electron Distributions) field analysis allowed a qualitative understanding of the structure-activity relationships. A representative compound N-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)-10H-phenothiazine-10-carboxamide (DT-PTZ-C) was able to inhibit completely oxidative damage brought about by two different procedures in organotypic hippocampal slice cultures, displaying a 30- to 100-fold higher potency than the standard vitamin E analogue, Trolox or edaravone. The compounds are novel, small, drug-like molecules of potential therapeutic use in neurodegenerative disorders and other conditions associated with oxidative stress.

Nitric oxide consumption through lipid peroxidation in brain cell suspensions and homogenates.

Mechanisms which inactivate NO (nitric oxide) are probably important in governing the physiological and pathological effects of this ubiquitous signalling molecule. Cells isolated from the cerebellum, a brain region rich in the NO signalling pathway, consume NO avidly. This property was preserved in brain homogenates and required both particulate and supernatant fractions. A purified fraction of the particulate component was rich in phospholipids, and NO consumption was inhibited by procedures that inhibited lipid peroxidation, namely a transition metal chelator, the vitamin E analogue Trolox and ascorbate oxidase. The requirement for the supernatant was accounted for by its content of ascorbate which catalyses metal-dependent lipid peroxidation. The NO-degrading activity of the homogenate was mimicked by a representative mixture of brain lipids together with ascorbate and, under these conditions, the lipids underwent peroxidation. In a suspension of cerebellar cells, there was a continuous low level of lipid peroxidation, and consumption of NO by the cells was decreased by approx. 50% by lipid-peroxidation inhibitors. Lipid peroxidation was also abolished when NO was supplied at a continuously low rate (approximately 100 nM/min), which explains why NO consumption by this process is saturable. Part of the activity remaining after the inhibition of lipid peroxidation was accounted for by contaminating red blood cells, but there was also another component whose activity was greatly enhanced when the cells were maintained under air-equilibrated conditions. A similar NO-consuming process was present in cerebellar glial cells grown in tissue culture but not in blood platelets or leucocytes, suggesting a specialized mechanism.