Christina Weinigel

Potent inhibition of human 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 by the anti-carcinogenic and anti-inflammatory agent embelin

Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) possesses anti-inflammatory and anti-carcinogenic properties in vivo, and these features have been related to interference with multiple targets including XIAPs, NFκB, STAT-3, Akt and mTOR. However, interference with these proteins requires relatively high concentrations of embelin (IC₅₀>4 μM) and cannot fully explain its bioactivity observed in several functional studies. Here we reveal human 5-lipoxygenase (5-LO) and microsomal prostaglandin E₂ synthase (mPGES)-1 as direct molecular targets of embelin. Thus, embelin potently suppressed the biosynthesis of eicosanoids by selective inhibition of 5-LO and mPGES-1 with IC₅₀=0.06 and 0.2 μM, respectively. In intact human polymorphonuclear leukocytes and monocytes, embelin consistently blocked the biosynthesis of various 5-LO products regardless of the stimulus (fMLP or A23187) with IC₅₀=0.8-2 μM. Neither the related human 12- and 15-LO nor the cyclooxygenases-1 and -2 or cytosolic phospholipase A₂ were significantly affected by 10 μM embelin. Inhibition of 5-LO and mPGES-1 by embelin was (I) essentially reversible after wash-out, (II) not impaired at higher substrate concentrations, (III) unaffected by inclusion of Triton X-100, and (IV) did not correlate to its proposed antioxidant properties. Docking simulations suggest concrete binding poses in the active sites of both 5-LO and mPGES-1. Because 5-LO- and mPGES-1-derived eicosanoids play roles in inflammation and cancer, the interference of embelin with these enzymes may contribute to its biological effects and suggests embelin as novel chemotype for development of dual 5-LO/mPGES-1 inhibitors.

Testosterone suppresses phospholipase D, causing sex differences in leukotriene biosynthesis in human monocytes

Sex disparities in inflammation have been reported, but the cellular and molecular basis for these discrepancies is unknown. Monocytes are central effector cells in immunity and possess high capacities to produce proinflammatory leukotrienes (LTs). Here, we investigated sex differences in the activation of 5‐lipoxygenase (5‐LO), the key enzyme in LT biosynthesis, in human peripheral monocytes. In cells from females, 5‐LO product formation was 1.8‐fold higher than in cells from males, as evaluated by HPLC. When female monocytes were resuspended in plasma from males, 5‐LO products were significantly lower than in female plasma. Interestingly, 5α‐dihydrotestosterone (5α‐DHT, 10 nM) repressed LT synthesis in female cells down to the levels observed in males, while estradiol (100 nM) was without effect, and progesterone (100 nM) caused only a slight inhibition. 5α‐DHT (10 nM) caused ERK phosphorylation and inhibition of phospholipase D (PLD), as evaluated by Western blot and measurement of PLD activity via radioenzymatic diacylglyceride (DAG) and nonradioactive choline assays. Accordingly, PLD activity and DAG formation were 1.4‐ to 1.8‐fold lower in male vs. female monocytes connected to increased ERK phosphorylation. Our data indicate that ERK activation by androgens in monocytes represses PLD activity, resulting in impaired 5‐LO product formation due to lack of activating DAGs.—Pergola, C., Rogge, A., Dodt, G., Northoff, H., Weinigel, C., Barz, D., Rådmark, O., Sautebin, L., Werz, O. Testosterone suppresses phospholipase D, causing sex differences in leukotriene biosynthesis in human monocytes. FASEB J. 25, 3377–3387 (2011). www.fasebj.org

Aminothiazole-Featured Pirinixic Acid Derivatives As Dual 5-Lipoxygenase and Microsomal Prostaglandin E2 Synthase-1 Inhibitors with Improved Potency and Efficiency in Vivo

Dual inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) is currently pursued as potential pharmacological strategy for treatment of inflammation and cancer. Here we present a series of 26 novel 2-aminothiazole-featured pirinixic acid derivatives as dual 5-LO/mPGES-1 inhibitors with improved potency (exemplified by compound 16 (2-[(4-chloro-6-{[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]amino}pyrimidin-2-yl)sulfanyl]octanoic acid) with IC50 = 0.3 and 0.4 μM, respectively) and bioactivity in vivo. Computational analysis presumes binding sites of 16 at the tip of the 5-LO catalytic domain and within a subpocket of the mPGES-1 active site. Compound 16 (10 μM) hardly suppressed cyclooxygenase (COX)-1/2 activities, failed to inhibit 12/15-LOs, and is devoid of radical scavenger properties. Finally, compound 16 reduced vascular permeability and inflammatory cell infiltration in a zymosan-induced mouse peritonitis model accompanied by impaired levels of cysteinyl-leukotrienes and prostaglandin E2. Together, 2-aminothiazole-featured pirinixic acids represent potent dual 5-LO/mPGES-1 inhibitors with an attractive pharmacological profile as anti-inflammatory drugs.

SAR Studies on Curcumin’s Pro-inflammatory Targets: Discovery of Prenylated Pyrazolocurcuminoids as Potent and Selective Novel Inhibitors of 5-Lipoxygenase

The anticarcinogenic and anti-inflammatory properties of curcumin have been extensively investigated, identifying prostaglandin E2 synthase (mPGES)-1 and 5-lipoxygenase (5-LO), key enzymes linking inflammation with cancer, as high affinity targets. A comparative structure-activity study revealed three modifications dissecting mPGES-1/5-LO inhibition, namely (i) truncation of the acidic, enolized dicarbonyl moiety and/or replacement by pyrazole, (ii) hydrogenation of the interaryl linker, and (iii) (dihydro)prenylation. The prenylated pyrazole analogue 11 selectively inhibited 5-LO, outperforming curcumin by a factor of up to 50, and impaired zymosan-induced mouse peritonitis along with reduced 5-LO product levels. Other pro-inflammatory targets of curcumin (i.e., mPGES-1, cyclooxygenases, 12/15-LOs, nuclear factor-κB, nuclear factor-erythroid 2-related factor-2, and signal transducer and activator of transcription 3) were hardly affected by 11. The strict structural requirements for mPGES-1 and 5-LO inhibition strongly suggest that specific interactions rather than redox or membrane effects underlie the inhibition of mPGES-1 and 5-LO by curcumin.

Design and synthesis of a second series of triazole-based compounds as potent dual mPGES-1 and 5-lipoxygenase inhibitors.

Microsomal prostaglandin E(2) synthase (mPGES)-1 and 5-lipoxygenase (5-LO) are pivotal enzymes in the biosynthesis of the pro-inflammatory PGE(2) and leukotrienes, respectively. The design and synthesis of a second series of mPGES-1 inhibitors based on a triazole scaffold are described. Our studies allowed us to draw a tentative SAR profile and to optimize this series with the identification of compounds 10, 11 and 14-15 which displayed potent mPGES-1 inhibition in a cell-free assay. In addition, compounds 5, 10, 12 and 14-16 also blocked 5-LO activity in cell-free and cell-based test systems, emerging as very promising candidates for the development of safer and more effective anti-inflammatory drugs.

Indirubin Core Structure of Glycogen Synthase Kinase-3 Inhibitors as Novel Chemotype for Intervention with 5-Lipoxygenase

The enzymes 5-lipoxygenase (5-LO) and glycogen synthase kinase (GSK)-3 represent promising drug targets in inflammation. We made use of the bisindole core of indirubin, present in GSK-3 inhibitors, to innovatively target 5-LO at the ATP-binding site for the design of dual 5-LO/GSK-3 inhibitors. Evaluation of substituted indirubin derivatives led to the identification of (3Z)-6-bromo-3-[(3E)-3-hydroxyiminoindolin-2-ylidene]indolin-2-one (15) as a potent, direct, and reversible 5-LO inhibitor (IC50 = 1.5 μM), with comparable cellular effectiveness on 5-LO and GSK-3. Together, we present indirubins as novel chemotypes for the development of 5-LO inhibitors, the interference with the ATP-binding site as a novel strategy for 5-LO targeting, and dual 5-LO/GSK-3 inhibition as an unconventional and promising concept for anti-inflammatory intervention.

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

An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

BACKGROUND Subcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists. METHODS FLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy. RESULTS 5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca(2+)-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB4. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP. CONCLUSION The cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging. GENERAL SIGNIFICANCE We present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.