The novel benzimidazole derivative BRP‐7 inhibits leukotriene biosynthesis in vitro and in vivo by targeting 5‐lipoxygenase‐activating protein (FLAP)

Abstract

BACKGROUND AND PURPOSELeukotrienes (LTs) are inflammatory mediators produced via the 5-lipoxygenase (5-LOX) pathway and are linked to diverse disorders, including asthma, allergic rhinitis and cardiovascular diseases. We recently identified the benzimidazole derivative BRP-7 as chemotype for anti-LT agents by virtual screening targeting 5-LOX-activating protein (FLAP). Here, we aimed to reveal the in vitro and in vivo pharmacology of BRP-7 as an inhibitor of LT biosynthesis. EXPERIMENTAL APPROACHWe analysed LT formation and performed mechanistic studies in human neutrophils and monocytes, in human whole blood (HWB) and in cell-free assays. The effectiveness of BRP-7 in vivo was evaluated in rat carrageenan-induced pleurisy and mouse zymosan-induced peritonitis. KEY RESULTSBRP-7 potently suppressed LT formation in neutrophils and monocytes and this was accompanied by impaired 5-LOX co-localization with FLAP. Neither the cellular viability nor the activity of 5-LOX in cell-free assays was affected by BRP-7, indicating that a functional FLAP is needed for BRP-7 to inhibit LTs, and FLAP bound to BRP-7 linked to a solid matrix. Compared with the FLAP inhibitor MK-886, BRP-7 did not significantly inhibit COX-1 or microsomal prostaglandin E2 synthase-1, implying the selectivity of BRP-7 for FLAP. Finally, BRP-7 was effective in HWB and impaired inflammation in vivo, in rat pleurisy and mouse peritonitis, along with reducing LT levels. CONCLUSIONS AND IMPLICATIONSBRP-7 potently suppresses LT biosynthesis by interacting with FLAP and exhibits anti-inflammatory effectiveness in vivo, with promising potential for further development. Abbreviations12-HHT, 12-hydroxy-5,8,10-heptadecatrienoic acid; 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; 5-LOX, 5-lipoxygenase; AA, arachidonic acid; cPLA2, cytosolic PLA2; cysLTs, cysteinyl LTs; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FLAP, fMLP,; hERG, human ether-a-go-go gene; HWB, human whole blood; IFM, immunofluorescence microscopy; mPGES-1, microsomal PGE2 synthase-1; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; PGC buffer, PBS pH 7.4 containing 1 mg·mL

Time‐resolvedin situassembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes

“…In fact, ample supply of exogenous arachidonic acid overcomes cellular 5-LO product synthesis inhibition by FLAP (25). Our data show that FLAP antagonists efficiently prevent the 5-LO/FLAP interaction, seemingly by interference with the adaptor functionality of arachidonic Figure 6.…”

Design, synthesis and evaluation of semi-synthetic triazole-containing caffeic acid analogues as 5-lipoxygenase inhibitors

“…Thus, in addition to the 5-LO activity assay in neutrophils and on the isolated enzyme, compound 10a, 15 and 16 were tested in a HEK cell system that stably expresses 5-LO with or without FLAP (Fig. 5) [18].…”

“…Two different HEK cell lines were tested (HEK_5-LO; HEK-5LO/ FLAP) as described by Pergola et al [18]. 1 Â 10 6 cells were suspended in 1 mL PGC buffer (PBS; 0.1% glucose, 1 mM CaCl 2 , preincubated with the test compound for 10 min at 37 C and subsequently stimulated by 2.5 mM Ca 2þ -ionophore A23187) plus 2 mM arachidonic acid for 10 min at 37 C. The reaction was stopped by 1 mL methanol, and the metabolites (all-trans isomers of LTB4 and 5-H(P)ETE) were extracted and analysed by HPLC as described above.…”

Novel series of benzoquinones with high potency against 5-lipoxygenase in human polymorphonuclear leukocytes

“…PMNL were either stimulated with the Ca 2þ -ionophore A23187 alone or together with 20 mM exogenous AA. Co-addition of AA allows circumventing the absolute need for endogenous substrate release by cPLA 2 and the AA transfer via the 5-LO-activating protein (FLAP) [7,22]. The 5-LO inhibitor 29 (N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea; zileuton) served as reference compound [23].…”