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

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.

Endogenous metabolites of vitamin E limit inflammation by targeting 5-lipoxygenase

“…To investigate the effect of LCMs on LTC 4 S activity, we used microsomal membranes from HEK-293 cells stably expressing LTC 4 S 70 . Membranes (2.5 µg total protein) were treated with LCMs for 10 min at 4 °C in potassium phosphate buffer (0.1 M, pH 7.4) containing 5 mM glutathione.…”

“…To investigate the effect of LCMs on sEH activity, we purified the human recombinant enzyme from sEH expressing Sf9 insect cells by benzylthio-sepharose affinity chromatography 70 . Sf9 cells were infected with a recombinant baculovirus (72 h) and sonicated (3 × 10 s, 4 °C) in lysis buffer (50 mM NaHPO 4 pH 8, 300 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM phenylmethansulfonyl fluoride, 10 µg/ml leupeptin, 60 µg/ml soybean trypsin inhibitor).…”

Adamantyl thioureas as soluble epoxide hydrolase inhibitors

“…Most of compounds reported as sEH inhibitors are 1,3-disubstituted ureas. 11–15 To our knowledge, only 10 thioureas have been reported as sEH inhibitors 16,17 compared to thousands of ureas. Thus, a systematic investigation of thioureas as sEH inhibitors is needed.…”

Novel Analgesics with Peripheral Targets

“…5-LOX inhibitor development has also been pursued, outputting compounds such as flavocoxid (discontinued after phase I) [533] and atreleuton (VIA-2291, discontinued after phase III) [534]. Diflapolin, a dual inhibitor of 5-LOX and soluble epoxide hydrolase, emerged as a promising anti-inflammatory tool [535]. LTB4, an end product of the LOX signaling pathway has been involved in inflammation [536] and has been shown to generate hyperalgesia in an intracutaneous injection model in humans [537].…”