A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors |
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Authors: | J Brian Jameson II Victor KenyonTheodore R Holman |
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Institution: | Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, CA 95064, USA |
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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). |
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Keywords: | Human 15-lipoxygenase-1 Human 15-lipoxygenase-2 High-throughput assay Inhibitor Allostery Mass spectroscopy |
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