title: 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.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.
authors.forename: J Brian
authors.initials: JB
authors.lastname: Jameson
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA.
authors.forename: Victor
authors.initials: V
authors.lastname: Kenyon
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA. Electronic address: tholman@chemistry.ucsc.edu.
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006801:Humans; D008041:Linoleic Acids / Q000737:chemistry; D016859:Lipoxygenase Inhibitors / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D015394:Molecular Structure; D013379:Substrate Specificity
chemical_list: D008041:Linoleic Acids; D016859:Lipoxygenase Inhibitors; C024348:13-hydroxy-9,11-octadecadienoic acid
keywords: Allostery; High-throughput assay; Human 15-lipoxygenase-1; Human 15-lipoxygenase-2; Inhibitor; Mass spectroscopy