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Pharmaceutical Research

, Volume 34, Issue 2, pp 378–393 | Cite as

Why Have Clinical Trials of Antioxidants to Prevent Neurodegeneration Failed? - A Cellular Investigation of Novel Phenothiazine-Type Antioxidants Reveals Competing Objectives for Pharmaceutical Neuroprotection

  • Maike J. Ohlow
  • Selina Sohre
  • Matthias Granold
  • Mathias Schreckenberger
  • Bernd Moosmann
Research Paper

Abstract

Purpose

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.

Methods

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.

Results

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.

Conclusions

Lipophilicity, radical stabilization and molecular weight appear to form an uneasy triangle, in which a slightly faulty selection may readily abolish neuroprotective activity.

KEY WORDS

neuroprotection oxidative stress phenothiazine protein oxidation structure-activity relationship 

Abbreviations

ABTS

2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)

ASC

Ascorbic acid; vitamin C

BCA

Bicinchoninic acid

BHT

Butylated hydroxytoluene

DCFA

2′,7′-Dichlorofluorescin diacetate

DMEM

Dulbecco’s modified Eagle medium

DNPH

2,4-Dinitrophenylhydrazine

EBS

Ebselen

EDA

Edaravone

FCS

Fetal calf serum

HT22

Glutamate-sensitive murine hippocampal cells

LUMOr

Lowest unoccupied molecular orbital of a radical

MTT

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetra-zolium bromide

NXY

NXY-059; disufenton sodium

PI

Propidium iodide

TBA

Thiobarbituric acid

TBARS

Thiobarbituric acid-reactive substances

TEAC

Trolox equivalent antioxidative capacity

TOC

α-Tocopherol; vitamin E

TROX

Trolox; 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

U74389G

Methyl tirilazad

Notes

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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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Institute for PathobiochemistryUniversity Medical Center of the Johannes Gutenberg UniversityMainzGermany
  2. 2.Department of Nuclear MedicineUniversity Medical Center of the Johannes Gutenberg UniversityMainzGermany

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