A High Throughput Screen Identifies Potent and Selective Inhibitors to Human Epithelial 15-Lipoxygenase-2

Abstract

Lipoxygenase (LOX) enzymes catalyze the hydroperoxidation of arachidonic acid and other polyunsaturated fatty acids to hydroxyeicosatetraenoic acids with varying positional specificity to yield important biological signaling molecules. Human epithelial 15­lipoxygenase­2 (15-LOX-2) is a highly specific LOX isozyme that is expressed in epithelial tissue and whose activity has been correlated with suppression of tumor growth in prostate and other epithelial derived cancers. Despite the potential utility of an inhibitor to probe the specific role of 15-LOX-2 in tumor progression, no such potent/specific 15­LOX­2 inhibitors have been reported to date. This study employs high throughput screening to identify two novel, specific 15­LOX­2 inhibitors. MLS000545091 is a mixed-type inhibitor of 15-LOX-2 with a Ki of 0.9+/−0.4 µM and has a 20-fold selectivity over 5-LOX, 12-LOX, 15-LOX-1, COX-1, and COX-2. MLS000536924 is a competitive inhibitor with a Ki of 2.5+/−0.5 µM and also possesses 20-fold selectivity toward 15-LOX-2 over the other oxygenases, listed above. Finally, neither compound possesses reductive activity towards the active-site ferrous ion.

Mutagenesis of Sequence Determinants of Truncated Porcine ALOX15 Induces Changes in the Reaction Specificity by Altering the Catalytic Mechanism of Initial Hydrogen Abstraction

“…Lipoxygenases adopt a two‐domain structure. The catalytic subunit contains the active site whereas a role on regulating catalytic activity and membrane‐binding has been attributed to the N‐terminal β‐barrel domain (C2‐domain) of several mammalian ALOX12, ALOX15, ALOX15B and ALOX5 orthologs. In all those LOXs isoforms truncation of the N‐terminal domain impairs catalytic activity but, interestingly, does not alter reaction specificity.…”

A high-throughput mass spectrometric assay for discovery of human lipoxygenase inhibitors and allosteric effectors

“…[14,19,20,29] Ranking the potency of these inhibitors is vital to understanding structure/activity relationships and thus optimizing inhibitor binding. However, several compounds were previously discovered with IC 50 values below the limit of the UV LOX assay (IC 50 <10 nM).…”

“…[13] Identifying inhibitors to these and other human LOXs has been an area of continued research. [14–20] The common method for discovering LOX inhibitors is the UV absorbance based assay, however, this method requires high nanomolar concentrations of LOX to produce measurable enzymatic rates. Thus, this method limits the lower range of inhibitor potency to greater than 10 nM, as has already been observed.…”

Inhibition of key enzymes in the inflammatory pathway by hybrid molecules of terpenes and synthetic drugs: In vitro and in silico studies

“…[22] However, in the in vitro LOX inhibition, only compound 15 showed the same activity (Table 1). [22] However, in the in vitro LOX inhibition, only compound 15 showed the same activity (Table 1).…”