A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors.

Abstract

Lipoxygenases (LOXs) regulate inflammation through the production of a variety of molecules whose specific downstream effects are not entirely understood due to the complexity of the inflammation pathway. The generation of these biomolecules can potentially be inhibited and/or allosterically regulated by small synthetic molecules. The current work describes the first mass spectrometric high-throughput method for identifying small molecule LOX inhibitors and LOX allosteric effectors that change the substrate preference of human lipoxygenase enzymes. Using a volatile buffer and an acid-labile detergent, enzymatic products can be directly detected using high-performance liquid chromatography-mass spectrometry (HPLC-MS) without the need for organic extraction. The method also reduces the required enzyme concentration compared with traditional ultraviolet (UV) absorbance methods by approximately 30-fold, allowing accurate binding affinity measurements for inhibitors with nanomolar affinity. The procedure was validated using known LOX inhibitors and the allosteric effector 13(S)-hydroxy-9Z,11E-octadecadienoic acid (13-HODE).

• Authors: J Brian Jameson 2nd, Victor Kenyon, Theodore R Holman
• Journal: Analytical biochemistry (Anal Biochem) Vol. 476 Pg. 45-50 (May 01 2015) ISSN: 1096-0309 [Electronic] United States
• PMID: 25712042 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• Linoleic Acids
• Lipoxygenase Inhibitors
• 13-hydroxy-9,11-octadecadienoic acid

Topics

• Chromatography, High Pressure Liquid
• Humans
• Linoleic Acids (chemistry)
• Lipoxygenase Inhibitors (chemistry)
• Mass Spectrometry (methods)
• Molecular Structure
• Substrate Specificity