Why Have Clinical Trials of Antioxidants to Prevent Neurodegeneration Failed? - A Cellular Investigation of Novel Phenothiazine-Type Antioxidants Reveals Competing Objectives for Pharmaceutical Neuroprotection
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Only a fraction of the currently established low-molecular weight antioxidants exhibit cytoprotective activity in living cells, which is considered a prerequisite for their potential clinical usefulness in Parkinson’s disease or stroke. Post hoc structure-activity relationship analyses have predicted that increased lipophilicity and enhanced radical stabilization could contribute to such cytoprotective activity.
We have synthesized a series of novel phenothiazine-type antioxidants exhibiting systematic variation in their lipophilicity and radical stabilization. Phenothiazine was chosen as lead structure for its superior activity at baseline. The novel compounds were evaluated for their neuroprotective potency in cell culture, and for their primary molecular targets.
Lipophilicity was associated with enhanced cytoprotective activity, but only to a certain threshold (logP ≈ 7). Benzannulation likewise produced improved cytoprotectants that exhibited very low EC50 values of ~8 nM in cultivated neuronal cells. Inhibition of global protein oxidation was the best molecular predictor of cytoprotective activity, followed by the inhibition of membrane protein autolysis. In contrast, the inhibition of lipid peroxidation in isolated brain lipids and the suppression of intracellular oxidant accumulation were poor predictors of cytoprotective activity, primarily as they misjudged the cellular advantage of high lipophilicity.
Lipophilicity, radical stabilization and molecular weight appear to form an uneasy triangle, in which a slightly faulty selection may readily abolish neuroprotective activity.
KEY WORDSneuroprotection oxidative stress phenothiazine protein oxidation structure-activity relationship
Ascorbic acid; vitamin C
Dulbecco’s modified Eagle medium
Fetal calf serum
Glutamate-sensitive murine hippocampal cells
Lowest unoccupied molecular orbital of a radical
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide
NXY-059; disufenton sodium
Thiobarbituric acid-reactive substances
Trolox equivalent antioxidative capacity
α-Tocopherol; vitamin E
Trolox; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
ACKNOWLEDGMENTS AND DISCLOSURES
The authors would like to thank Dr. James V. Crivello for his generous contribution of compounds 8, 9 and 10. The authors declare that they have no conflicts of interest. This work was supported by the Neuro Graduate School and the Interdisciplinary Research Centre for Neurosciences of the University of Mainz, which are non-profit entities that had no role in the design, execution, interpretation, or publication of this study.
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