title: A potent and selective inhibitor targeting human and murine 12/15-LOX.
abstract: Human reticulocyte 12/15-lipoxygenase (h12/15-LOX) is a lipid-oxidizing enzyme that can directly oxidize lipid membranes in the absence of a phospholipase, leading to a direct attack on organelles, such as the mitochondria. This cytotoxic activity of h12/15-LOX is up-regulated in neurons and endothelial cells after a stroke and thought to contribute to both neuronal cell death and blood-brain barrier leakage. The discovery of inhibitors that selectively target recombinant h12/15-LOX in vitro, as well as possessing activity against the murine ortholog ex vivo, could potentially support a novel therapeutic strategy for the treatment of stroke. Herein, we report a new family of inhibitors discovered in a High Throughput Screen (HTS) that are selective and potent against recombinant h12/15-LOX and cellular mouse 12/15-LOX (m12/15-LOX). MLS000099089 (compound 99089), the parent molecule, exhibits an IC50 potency of 3.4±0.5 μM against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 μM in a mouse neuronal cell line, HT-22. Compound 99089 displays greater than 30-fold selectivity versus h5-LOX and COX-2, 15-fold versus h15-LOX-2 and 10-fold versus h12-LOX, when tested at 20 μM inhibitor concentration. Steady-state inhibition kinetics reveals that the mode of inhibition of 99089 against h12/15-LOX is that of a mixed inhibitor with a Kic of 1.0±0.08 μM and a Kiu of 6.0±3.3 μM. These data indicate that 99089 and related derivatives may serve as a starting point for the development of anti-stroke therapeutics due to their ability to selectively target h12/15-LOX in vitro and m12/15-LOX ex vivo.
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95060, United States.
authors.forename: Michelle M
authors.initials: MM
authors.lastname: Armstrong
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95060, United States.
authors.forename: Cody J
authors.initials: CJ
authors.lastname: Freedman
authors.affiliation: Neuroprotection Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, United States.
authors.forename: Joo Eun
authors.initials: JE
authors.lastname: Jung
authors.affiliation: Neuroprotection Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, United States.
authors.forename: Yi
authors.initials: Y
authors.lastname: Zheng
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143, United States.
authors.forename: Chakrapani
authors.initials: C
authors.lastname: Kalyanaraman
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, CA 94143, United States.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jacobson
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892, United States.
authors.forename: Anton
authors.initials: A
authors.lastname: Simeonov
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892, United States.
authors.forename: David J
authors.initials: DJ
authors.lastname: Maloney
authors.affiliation: Neuroprotection Research Laboratory, Department of Radiology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, United States.
authors.forename: Klaus
authors.initials: K
authors.lastname: van Leyen
authors.affiliation: National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, MSC 3370, Bethesda, MD 20892, United States.
authors.forename: Ajit
authors.initials: A
authors.lastname: Jadhav
authors.affiliation: Chemistry and Biochemistry Department, University of California, Santa Cruz, CA 95060, United States. Electronic address: holman@ucsc.edu.
authors.forename: Theodore R
authors.initials: TR
authors.lastname: Holman
mesh_terms: D000818:Animals; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D057166:High-Throughput Screening Assays; D006801:Humans; D016859:Lipoxygenase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: C475064:12-15-lipoxygenase; D016859:Lipoxygenase Inhibitors; D011994:Recombinant Proteins; D001092:Arachidonate 12-Lipoxygenase; D001093:Arachidonate 15-Lipoxygenase
keywords: High-throughput; Human; Inhibitor; Lipoxygenase; Murine; Selective