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