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.

Keywords: High-throughput; Human; Inhibitor; Lipoxygenase; Murine; Selective.

Figure 1

Relevant h12/15-LOX inhibitors.

Figure 1

Relevant h12/15-LOX inhibitors.

Figure 2

Structure and potency of the novel h12/15-LOX inhibitor (error in parentheses).

Figure 3

Titration of 99089 in HT-22 cells, with increasing cell survival. ML351 was used as a positive control at 10 µM.

Figure 4

Selectivity potency (µM) against h12-LOX, h15-LOX-2 and 5-LOX with 99089. Three inhibitor concentrations (20 µM, 25 µM, 35 µM) were screened against h12-LOX and h15-LOX-2, and the IC₅₀ value averaged. For h5-LOX and COX-2 we only screened at 20 µM, due to their low potency and the solubility problems of 99089 above 35 µM.

Figure 5

Steady-state inhibition kinetics for the determination of K_ic and K_iu of h12/15-LOX and 99089. (A) Dixon plot of the primary data of h12/15-LOX and 99089. The substrate concentrations are 2 µM (open triangles), 4 µM (closed squares), 9 µM (open circles), 15 µM (closed diamonds). (B) The Dixon replot of slope versus [Inhibitor] in µM yielded a K_ic of 1.0 (0.08) µM and a K_iu of 6.0 (3) µM.

Figure 6

99089 docking pose in the active site of h12/15-LOX. Carbon atoms of the protein and ligand are shown in grey and green, respectively. Nitrogen, oxygen, hydrogen atoms are blue, red and white, respectively. The ferric ion is shown as an orange sphere. 99089 is shown in ball-and-stick representation.