A potent and selective inhibitor targeting human and murine 12/15-LOX

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 mu M against h12/15-LOX in vitro and an ex vivo IC50 potency of approximately 10 mu M 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 mu M 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 K-ic of 1.0 +/- 0.08 lM and a K-iu of 6.0 +/- 3.3 mu M. 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. (C) 2016 Elsevier Ltd. All rights reserved.