Jana Gerstmeier

Identification of novel benzimidazole derivatives as inhibitors of leukotriene biosynthesis by virtual screening targeting 5-lipoxygenase-activating protein (FLAP).

Pharmacological suppression of leukotriene biosynthesis by 5-lipoxygenase (5-LO)-activating protein (FLAP) inhibitors is a promising strategy to intervene with inflammatory, allergic and cardiovascular diseases. Virtual screening targeting FLAP based on a combined ligand- and structure-based pharmacophore model led to the identification of 1-(2-chlorobenzyl)-2-(1-(4-isobutylphenyl)ethyl)-1H-benzimidazole (7) as developable candidate. Compound 7 potently suppressed leukotriene formation in intact neutrophils (IC(50)=0.31 μM) but essentially failed to directly inhibit 5-LO suggesting that interaction with FLAP causes inhibition of leukotriene synthesis. For structural optimization, a series of 46 benzimidazole-based derivatives of 7 were synthesized leading to more potent analogues (70-72, 82) with IC(50)=0.12-0.19 μM in intact neutrophils. Together, our results disclose the benzimidazole scaffold bearing an ibuprofen fingerprint as a new chemotype for further development of anti-leukotriene agents.

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

Leukotrienes (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.

Time-resolved in situ assembly of the leukotriene-synthetic 5-lipoxygenase/5-lipoxygenase-activating protein complex in blood leukocytes

5‐Lipoxygenase (5‐LO) catalyzes the initial steps in the biosynthesis of proinflammatory leukotrienes. Upon cell activation, 5‐LO translocates to the nuclear membrane where arachidonic acid is transferred by 5‐LO‐activating protein (FLAP) to 5‐LO for metabolism. Although previous data indicate association of 5‐LO with FLAP, the in situ assembly of native 5‐LO/FLAP complexes remains elusive. Here, we show time‐resolved 5‐LO/FLAP colocalization by immunofluorescence microscopy and in situ 5‐LO/FLAP interaction by proximity ligation assay at the nuclear membrane of Ca2+‐ionophore A23187‐activated human monocytes and neutrophils in relation to 5‐LO activity. Although 5‐LO translocation and product formation is completed within 1.5‐3 min, 5‐LO/FLAP interaction is delayed and proceeds up to 30 min. Though monocytes and neutrophils contain comparable amounts of 5‐LO protein, neutrophils produce 3‐5 times higher levels of 5‐LO products due to prolonged activity, accompanied by delayed 5‐LO nuclear membrane translocation. Arachidonic acid seemingly acts as adaptor for 5‐LO/FLAP assembly, whereas FLAP inhibitors (MK886, 100 nM; BAY X 1005, 3 μM) disrupt the complex. We conclude that FLAP may regulate 5‐LO activity in 2 ways: first by inducing an initial flexible association for efficient 5‐LO product synthesis, followed by the formation of a tight 5‐LO/FLAP complex that terminates 5‐LO activity.—Gerstmeier, J., Weinigel, C., Rummler, S., Rådmark, O., Werz, O., Garscha, U. Time‐resolved in situ assembly of the leukotriene‐synthetic 5‐lipoxygenase/5‐lipoxygenase‐activating protein complex in blood leukocytes. FASEB J. 30, 276‐285 (2016). www.fasebj.org

4,5-Diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP)

In this article, we report novel leukotriene (LT) biosynthesis inhibitors that may target 5-lipoxygenase-activating protein (FLAP) based on the previously identified isoxazole derivative (8). The design and synthesis was directed towards a subset of 4,5-diaryl-isoxazole-3-carboxylic acid derivatives as LT biosynthesis inhibitors. Biological evaluation disclosed a new skeleton of potential anti-inflammatory agents, exemplified by 39 and 40, which potently inhibit cellular 5-LO product synthesis (IC50 = 0.24 μM, each) seemingly by targeting FLAP with weak inhibition on 5-LO (IC50 ≥ 8 μM). Docking studies and molecular dynamic simulations with 5-LO and FLAP provide valuable insights into potential binding modes of the inhibitors. Together, these diaryl-isoxazol-3-carboxylic acids may possess potential as leads for development of effective anti-inflammatory drugs through inhibition of LT biosynthesis.

5-Lipoxygenase-activating protein rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation

Leukotrienes (LTs) are proinflammatory lipid mediators formed from arachidonic acid in a 2‐step reaction catalyzed by 5‐lipoxygenase (5‐LOX) requiring the formation of 5‐HPETE [5(S)‐hydroperoxy‐6‐trans‐8,11,14‐cis‐eicosatetraenoic acid] and its subsequent transformation to LTA4. 5‐LOX is thought to receive arachidonic acid from the nuclear membrane‐embedded 5‐LOX‐activating protein (FLAP). The crystal structure of 5‐LOX revealed an active site concealed by F177 and Y181 (FY cork). We examined the influence of the FY cork on 5‐LOX activity and membrane binding in HEK293 cells in the absence and presence of FLAP. Uncapping the 5‐LOX active site by mutation of F177 and/or Y181 to alanine (5‐LOX‐F177A, 5‐LOX‐Y181A, 5‐LOX‐F177/Y181A) resulted in delayed and diminished 5‐LOX membrane association in A23187‐stimulated cells. For 5‐LOX‐F177A and 5‐LOX‐F177/Y181A, formation of 5‐LOX products was dramatically reduced relative to 5‐LOX‐wild type (wt). Strikingly, coexpression of FLAP in A23187‐activated HEK293 cells effectively restored formation of 5‐H(p)ETE (5‐hydroxy‐ and 5‐peroxy‐6‐trans‐8,11,14‐cis‐eicosatetraenoic acid) by these same 5‐LOX mutants (ã60–70% 5‐LOX‐wt levels) but not of LTA4 hydrolysis products. Yet 5‐LOX‐Y181A generated 5‐H(p)ETE at levels comparable to 5‐LOX‐wt but reduced LTA4 hydrolysis products. Coexpression of FLAP partially restored LTA4 hydrolysis product formation by 5‐LOX‐Y181A. Together, the data suggest that the concealed FY cork impacts membrane association and that FLAP may help shield an uncapped active site.—Gerstmeier, J., Newcomer, M. E., Dennhardt, S., Romp, E., Fischer, J., Werz, O., Garscha, U. 5‐Lipoxygenase‐activating protein rescues activity of 5‐lipoxygenase mutations that delay nuclear membrane association and disrupt product formation. FASEB J. 30, 1892–1900 (2016). www.fasebj.org

Chromatographic separation and biological evaluation of benzimidazole derivative enantiomers as inhibitors of leukotriene biosynthesis

For an explicit analysis of the chirality on the effectiveness of a recently identified racemic benzimidazole derivative (BRP7) as inhibitor of leukotriene biosynthesis, we optimized a HPLC-based chiral chromatographic method enabling the quantitative isolation of its enantiomers in sufficient amount to carry out biological investigations. The use of a Lux Amylose-2 column revealed especially profitable to fulfil our task. Indeed, the employment of the amylose-based chiral stationary phase (CSP) in combination with a n-hexane/EtOH/DEA - 99/1/02 (v/v/v) mobile phase allowed getting the enantiomeric peaks fully resolved (α=1.80, RS=2.39). Four consecutive injections repeated at 1-min intervals produced overloaded peaks with a very limited level of isomeric contamination. This procedure allowed the isolation of ca. 20mg of each enantiomer, with enantiomeric excess higher than 99% and 95% for the (S)- and the (R)-isomer, respectively. The enantiomeric elution order was established using synthetic reference compounds of lower enantiomeric excess values. The biological evaluation of the purified individual enantiomers revealed no significant difference in terms of their IC50 values with respect to the previously investigated racemic BRP7: 0.18μM for the (R)-enantiomer (R(2)=0.999) and 0.26μM for the (S)-enantiomer (R(2)=0.986).

BRP-187: A potent inhibitor of leukotriene biosynthesis that acts through impeding the dynamic 5-lipoxygenase/5-lipoxygenase-activating protein (FLAP) complex assembly

The pro-inflammatory leukotrienes (LTs) are formed from arachidonic acid (AA) in activated leukocytes, where 5-lipoxygenase (5-LO) translocates to the nuclear envelope to assemble a functional complex with the integral nuclear membrane protein 5-LO-activating protein (FLAP). FLAP, a MAPEG family member, facilitates AA transfer to 5-LO for efficient conversion, and LT biosynthesis critically depends on FLAP. Here we show that the novel LT biosynthesis inhibitor BRP-187 prevents the 5-LO/FLAP interaction at the nuclear envelope of human leukocytes without blocking 5-LO nuclear redistribution. BRP-187 inhibited 5-LO product formation in human monocytes and polymorphonuclear leukocytes stimulated by lipopolysaccharide plus N-formyl-methionyl-leucyl-phenylalanine (IC50=7-10nM), and upon activation by ionophore A23187 (IC50=10-60nM). Excess of exogenous AA markedly impaired the potency of BRP-187. Direct 5-LO inhibition in cell-free assays was evident only at >35-fold higher concentrations, which was reversible and not improved under reducing conditions. BRP-187 prevented A23187-induced 5-LO/FLAP complex assembly in leukocytes but failed to block 5-LO nuclear translocation, features that were shared with the FLAP inhibitor MK886. Whereas AA release, cyclooxygenases and related LOs were unaffected, BRP-187 also potently inhibited microsomal prostaglandin E2 synthase-1 (IC50=0.2μM), another MAPEG member. In vivo, BRP-187 (10mg/kg) exhibited significant effectiveness in zymosan-induced murine peritonitis, suppressing LT levels in peritoneal exudates as well as vascular permeability and neutrophil infiltration. Together, BRP-187 potently inhibits LT biosynthesis in vitro and in vivo, which seemingly is caused by preventing the 5-LO/FLAP complex assembly and warrants further preclinical evaluation.

Androgen-mediated sex bias impairs efficiency of leukotriene biosynthesis inhibitors in males

Proinflammatory leukotrienes (LTs) are produced by 5-lipoxygenase (5-LO) aided by 5-LO–activating protein (FLAP). LT biosynthesis inhibitors are currently under clinical investigation as treatments for respiratory and cardiovascular diseases. Here, we have revealed a sex bias in the efficiency of clinically relevant LT biosynthesis inhibitors, showing that their effects are superior in females. We found that androgens cause these sex differences by impeding the LT-biosynthetic 5-LO/FLAP complex assembly. Lower doses of the FLAP inhibitor MK886 were required to reduce LTB4 levels in exudates of female versus male mice and rats. Following platelet-activating factor–induced shock, MK886 increased survival exclusively in female mice, and this effect was abolished by testosterone administration. FLAP inhibitors and the novel-type 5-LO inhibitors licofelone and sulindac sulfide exhibited higher potencies in human blood from females, and bioactive 5-LO/FLAP complexes were formed in female, but not male, human and murine leukocytes. Supplementation of female blood or leukocytes with 5&agr;-dihydrotestosterone abolished the observed sex differences. Our data suggest that females may benefit from anti-LT therapy to a greater extent than males, prompting consideration of sex issues in LT modifier development.

Novel leukotriene biosynthesis inhibitors (2012-2016) as anti-inflammatory agents

ABSTRACT Introduction: Leukotrienes (LTs) are lipid mediators produced from arachidonic acid with a broad variety of bioactivities in allergy and inflammation. The biosynthesis of LTs mainly involves 5-lipoxygenase (5-LO) and its 5-lipoxygenase-activating protein (FLAP), LTA4 hydrolase and LTC4 synthase that all may represent potential targets for LT biosynthesis inhibitors. Areas covered: We introduce the LT biosynthetic pathway and its cellular regulation, the diverse biological actions of LTs and their receptors, and we briefly describe the pharmacological strategies for suppression of LT formation as well as the classes of current LT biosynthesis inhibitors. The main focus is placed on the comprehensive discussion of recently reported inhibitors of 5-LO, FLAP, LTA4 hydrolase and LTC4 synthase, based on literature search (PubMed and Thomson Innovation Patents Searches), covering 2012–2016. Expert opinion: Although many new series of 5-LO inhibitors have been presented without patenting, essentially by academia, novel FLAP inhibitors (many patented) are most advanced in clinical development and are apparently the focus of pharmaceutical companies. Only few novel inhibitors of LTA4 hydrolase and LTC4 synthase were reported. Major issues in the development of LT synthesis inhibitors are related to loss of potency in biological relevant environment, poor pharmacokinetics, lack of oral efficacy, and side effects.

Characterization of the interaction of human 5-lipoxygenase with its activating protein FLAP

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.