Pharmacological profile and efficiency in vivo of diflapolin, the first dual inhibitor of 5-lipoxygenase-activating protein and soluble epoxide hydrolase

Ulrike Garscha,Jana Gerstmeier,Simona Pace,Markus Werner,Gerhard K Scriba,Lidia Sautebin,Stefanie Liening,Christina Weinigel,Silke Rummler,Antonietta Rossi,Erik Romp,Veronika Temml,Heiner Atze,Stefanie König,Sandra Wittmann,Daniela Schuster,Oliver Werz

doi:10.1038/s41598-017-09795-w

Abstract

Arachidonic acid (AA) is metabolized to diverse bioactive lipid mediators. Whereas the 5-lipoxygenase-activating protein (FLAP) facilitates AA conversion by 5-lipoxygenase (5-LOX) to pro-inflammatory leukotrienes (LTs), the soluble epoxide hydrolase (sEH) degrades anti-inflammatory epoxyeicosatrienoic acids (EETs). Accordingly, dual FLAP/sEH inhibition might be advantageous drugs for intervention of inflammation. We present the in vivo pharmacological profile and efficiency of N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N′-(3,4-dichlorophenyl)urea (diflapolin) that dually targets FLAP and sEH. Diflapolin inhibited 5-LOX product formation in intact human monocytes and neutrophils with IC50 = 30 and 170 nM, respectively, and suppressed the activity of isolated sEH (IC50 = 20 nM). Characteristic for FLAP inhibitors, diflapolin (I) failed to inhibit isolated 5-LOX, (II) blocked 5-LOX product formation in HEK cells only when 5-LOX/FLAP was co-expressed, (III) lost potency in intact cells when exogenous AA was supplied, and (IV) prevented 5-LOX/FLAP complex assembly in leukocytes. Diflapolin showed target specificity, as other enzymes related to AA metabolism (i.e., COX1/2, 12/15-LOX, LTA4H, LTC4S, mPGES1, and cPLA2) were not inhibited. In the zymosan-induced mouse peritonitis model, diflapolin impaired vascular permeability, inhibited cysteinyl-LTs and LTB4 formation, and suppressed neutrophil infiltration. Diflapolin is a highly active dual FLAP/sEH inhibitor in vitro and in vivo with target specificity to treat inflammation-related diseases.

Highlights

The arachidonic acid (AA) cascade plays a central role in the biosynthesis of lipid mediators (LMs) with pro-inflammatory and anti-inflammatory properties[1]
Based on a pharmacophore-based virtual screening campaign, diflapolin was identified as most potent agent out of 20 hit compounds that dually inhibited FLAP and soluble epoxide hydrolase (sEH) in simple screening assays[15]
A typical feature of FLAP inhibitors is their loss of efficiency, when cells are stimulated for 5-LOX product formation in the presence of exogenous AA, since (I) FLAP inhibitors compete with AA binding within the active site of FLAP26, and (II) ample AA supply may circumvent the requirement of FLAP for cellular 5-LOX product formation[27, 28]

Summary

Introduction

The arachidonic acid (AA) cascade plays a central role in the biosynthesis of lipid mediators (LMs) with pro-inflammatory and anti-inflammatory properties[1]. 15) as the first dual inhibitor of 5-lipoxygenase-activating protein (FLAP) and sEH by using a pharmacophore-based virtual screening[15] It was recently reported that co-administration of a sEH inhibitor with a FLAP inhibitor enhanced the anti-inflammatory activities in a murine model[21], whereas sole inhibition of sEH lead to albuminuria[22] These data support the development of dual FLAP/sEH inhibitors to achieve better therapeutic effects due to simultaneous suppression of pro-inflammatory LTs and DiHETrEs but maintaining anti-inflammatory EETs. Here, we present the molecular pharmacological profile and the effectiveness of the first dual FLAP/sEH inhibitor diflapolin[15] using cell-free and cell-based analysis of biosynthetic pathways of the AA cascade as well as animal models of inflammation. We find that diflapolin is target-specific for sEH and FLAP with strong potencies and represents a highly effective anti-inflammatory compound

Methods

Results

Conclusion