Leukotriene Biosynthesis Inhibitor Research Articles

Cysteinyl leukotriene-like metabolites are generated in retinal pigment epithelial cells through glutathionylation/reduction of an oxidatively truncated fragment of arachidonate

γ-Hydroxyalkenals, 4-hydroxynonenal (HNE) and phospholipid esters of 4-hydroxy-8-oxooctenoic acid (HOOA-PL), are produced from the alkyl and carboxyl termini of arachidonyl phospholipids by radical-induced oxidative cleavage. Metabolism of HNE by Michael addition of glutathione (GSH) followed by reduction of the aldehyde carbonyl produces a GSH derivative of 1,4-dihydroxynonane (DHN)-GSH. Analogous biochemistry was anticipated to produce a GSH derivative of 5,8-dihydroxyoctanoic acid (DHOA-GSH) that has structural and functional similarity to the cysteinyl leukotriene (LT)C4. We now report that exposure of human retinal pigment epithelial cells to CoCl2, an in vitro model of hypoxia-induced oxidative stress, generates DHOA-GSH and two products of its peptidolysis, DHOA-CysGly and DHOA-Cys that resemble LTD4 and LTE4. Identification of these metabolites was confirmed by unambiguous chemical syntheses that also provided a heavy isotope labeled quantitative standard 13C215N-DHOA-GSH. The availability of pure samples of these arachidonate metabolites will enable assessment of their biological activities, and testing the hypothesis that øLTs promote pathological inflammation by serving as LT receptor agonists. Because LT biosynthetic enzymes, e.g., 5-lipoxygenase, are not involved in the generation of øLTs in vivo, inhibitors of LT biosynthesis, e.g., Zileuton, are not expected to prevent the generation of øLTs. On the other hand, if øLTs are leukotriene receptor agonists, then the therapeutic effects of leukotriene receptor antagonist drugs, e.g., Montelukast, may include inhibition not only of LT-induced but also øLT-induced LT receptor activation and signaling.

Ethoxy acetalated dextran nanoparticles for drug delivery: A comparative study of formulation methods

Dextran-based polymers, such as ethoxy acetalated dextran (Ace-DEX), are increasingly becoming the focus of research as they offer great potential for the development of polymer-based nanoparticles as drug delivery vehicles. Their major advantages are the facile synthesis, straightforward particle preparation and the pH-dependent degradation of the particles that can be fine-tuned by the degree of acetalation of the polymer. In this study we have shown that Ace-DEX can not only compete against the commonly used and FDA-approved polymer poly(lactic-co-glycolic acid) (PLGA), but even has the potential to outperform it in its encapsulation properties, e.g., for the herein used anti-inflammatory leukotriene biosynthesis inhibitor BRP-187. We used three different methods (microfluidics, batch nanoprecipitation and emulsion solvent evaporation) for the preparation of BRP-187-loaded Ace-DEX nanoparticles to investigate the influence of the formulation technique on the physicochemical properties of the particles. Finally, we evaluated which production method offers the greatest potential for achieving the demands for a successful translation from research into pharmaceutical production by fulfilling the basic requirements, such as reaching a high loading capacity of the particles and excellent reproducibility while being simple and affordable.

Identification of 2-Aminoacyl-1,3,4-thiadiazoles as Prostaglandin E2 and Leukotriene Biosynthesis Inhibitors.

The application of a multi-step scientific workflow revealed an unprecedented class of PGE2/leukotriene biosynthesis inhibitors with in vivo activity. Specifically, starting from a combinatorial virtual library of ∼4.2 × 105 molecules, a small set of compounds was identified for the synthesis. Among these, four novel 2-aminoacyl-1,3,4-thiadiazole derivatives (3, 6, 7, and 9) displayed marked anti-inflammatory properties in vitro by strongly inhibiting PGE2 biosynthesis, with IC50 values in the nanomolar range. The hit compounds also efficiently interfered with leukotriene biosynthesis in cell-based systems and modulated IL-6 and PGE2 biosynthesis in a lipopolysaccharide-stimulated J774A.1 macrophage cell line. The most promising compound 3 showed prominent in vivo anti-inflammatory activity in a mouse model, with efficacy comparable to that of dexamethasone, attenuating zymosan-induced leukocyte migration in mouse peritoneum with considerable modulation of the levels of typical pro-/anti-inflammatory cytokines.

Structure-Activity Relationship Studies of New Sinapic Acid Phenethyl Ester Analogues Targeting the Biosynthesis of 5-Lipoxygenase Products: The Role of Phenolic Moiety, Ester Function, and Bioisosterism.

Sinapic acid is found in many edible plants and fruits, such as rapeseed, where it is the predominant phenolic compound. New sinapic acid phenethyl ester (SAPE) analogues were synthesized and screened as inhibitors of the biosynthesis of 5-lipoxygenase (5-LO) in stimulated HEK293 cells and polymorphonuclear leukocytes (PMNL). Inhibition of leukotriene biosynthesis catalyzed by 5-LO is a validated therapeutic strategy against certain inflammatory diseases and allergies. Unfortunately, the only inhibitor approved to date has limited clinical use because of its poor pharmacokinetic profile and liver toxicity. With the new analogues synthesized in this study, the role of the phenolic moiety, ester function, and bioisosterism was investigated. Several of the 34 compounds inhibited the biosynthesis of 5-LO products, and 20 compounds were 2-11 times more potent than zileuton in PMNL, which are important producers of 5-LO products. Compounds 5i (IC50: 0.20 μM), 5l (IC50: 0.20 μM), and 5o (IC50: 0.21 μM) bearing 4-trifluoromethyl, methyl, or methoxy substituent at meta-position of the phenethyl moiety were 1.5 and 11.5 times more potent than SAPE (IC50: 0.30 μM) and zileuton (IC50: 2.31 μM), respectively. Additionally, compound 9 (IC50: 0.27 μM), which was obtained after acetylation of the 4-hydroxyl of SAPE, was equivalent to SAPE and 8 times more active than zileuton. Furthermore, compound 20b (IC50: 0.27 μM) obtained after the bioisosteric replacement of the ester function of SAPE by the 1,2,4-oxadiazole heterocycle was equivalent to SAPE and 8 times more active than zileuton. Thus, this study provides a basis for the rational design of new molecules that could be developed further as anti 5-LO therapeutics.

5α-dihydrotestosterone abrogates sex bias in asthma like features in the mouse

Androgen levels inversely correlate with the incidence, susceptibility and severity of asthma. However, whether male sex hormones such as 5α-dihydrotestosterone (DHT) have beneficial effects on asthma symptoms and/or could affect asthma susceptibility have not been investigated. DHT administration to female mice, during the sensitization phase, abrogates the sex bias in bronchial hyperreactivity. This effect correlates with inhibition of leukotriene biosynthesis in the lung. DHT significantly inhibits also other asthma-like features such as airway hyperplasia and mucus production in sensitized female mice. Conversely, DHT does not affect plasma IgE levels as well as CD3+CD4+ IL-4+ cell and IgE+c-Kit+ cell infiltration within the lung but prevents pulmonary mast cell activation. The in vitro study on RBL‐2H3 cells confirms that DHT inhibits mast cell degranulation. In conclusion, our data demonstrate that immunomodulatory effects of DHT on mast cell activation prevent the translation of allergen sensitization into clinical manifestation of asthma.

Phenolic acid phenethylesters and their corresponding ketones: Inhibition of 5-lipoxygenase and stability in human blood and HepaRG cells.

5‐lipoxygenase (5‐LO) catalyzes the biosynthesis of leukotrienes, potent lipid mediators involved in inflammatory diseases, and both 5‐LO and the leukotrienes are validated therapeutic targets. Caffeic acid phenethyl ester (CAPE) is an effective inhibitor of 5‐LO and leukotriene biosynthesis but is susceptible to hydrolysis by esterases. In this study a number of CAPE analogues were synthesized with modifications to the caffeoyl moiety and the replacement of the ester linkage with a ketone. Several new molecules showed better inhibition of leukotriene biosynthesis than CAPE in isolated human neutrophils and in whole blood with IC50 values in the nanomolar (290‐520 nmol/L) and low micromolar (1.0‐2.3 µmol/L) ranges, respectively. Sinapic acid and 2,5‐dihydroxy derivatives were more stable than CAPE in whole blood, and ketone analogues were degraded more slowly in HepaRG hepatocyte cultures than esters. All compounds underwent modification consistent with glucuronidation in HepaRG cultures as determined using LC‐MS/MS analysis, though the modified sinapoyl ketone (10) retained 50% of its inhibitory activity after up to one hour of incubation. This study has identified at least one CAPE analogue, compound 10, that shows favorable properties that warrant further in vivo investigation as an antiinflammatory compound.

Abstract 1275: MK591 (Quiflapon) downregulates c-Myc oncogenic signaling and induces apoptosis in enzalutamide-resistant prostate cancer cells

Abstract Background: Enzalutamide is an FDA-approved drug commonly prescribed for advanced prostate cancer. Enzalutamide slows down prostate tumor growth but resistant disease invariably develops which is incurable primarily because currently available therapies cannot effectively kill enzalutamide-resistant prostate cancer cells. Mechanism(s) behind development of enzalutamide-resistance is not properly understood, though overactivation of c-Myc has been found to be a common event which plays an important role in the maintenance and progression of ERPC phenotype. However, direct-targeting of c-Myc poses special problem because of its non-enzymatic nature and certain amount of c-Myc activity is needed by non-cancer cells as well. Thus, c-Myc has emerged as an elusive target which needs to be managed by novel agents and strategies in a cancer-specific way for better control of ERPC. Methods: We addressed this problem by treating ERPC cells with a variety of cell survival and apoptosis-regulating agents followed by measurement of cell viability, and by analysis of the mRNA and protein levels of c-Myc. Gene expression was analyzed by Illumina Hi-Seq whole genome gene-expression array. Expression of c-Myc was confirmed by RT-PCR and Western blot. Apoptosis was measured by annexin-V binding, PARP-cleavage, and by detecting degradation of chromatin-DNA to nucleosomes. Transcriptional activity of c-Myc was analyzed by nuclear accumulation, DNA-binding, luciferase-reporter assays and expression of c-Myc-target genes. Results: We found that MK591, a leukotriene biosynthesis inhibitor, dramatically downregulates c-Myc in ERPC cells as revealed by RT-PCR, and Western blot as well as by the reduction in nuclear-accumulation and DNA-binding activities of c-Myc. Treatment with MK591 decreased the Myc-driven E-box-luciferase reporter activity, and substantially reduced the expression of c-Myc target genes (Cyclin D1, CDK4, survivin, Aurora kinase). Moreover, MK591 effectively blocked in vitro invasion and soft-agar colony-formation by ERPC cells. Interestingly, while MK591 strongly inhibits c-Myc function and kills ERPC cells via caspase-mediated apoptosis, it does not inhibit the basal c-Myc function or the viability of non-cancer cells, such as human foreskin fibroblasts (HFF). Conclusion: Our findings indicate that the expression and oncogenic-function of c-Myc in ERPC cells are severely downregulated by MK591, and suggest that MK591 may turn out to be a suitable new agent to treat advanced, aggressive prostate cancers which are resistant to enzalutamide therapy. Citation Format: Jitender Monga, Ajay Bharathan, Dhatchayini Subramani, Jagadananda Ghosh. MK591 (Quiflapon) downregulates c-Myc oncogenic signaling and induces apoptosis in enzalutamide-resistant prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1275.

Learning Impairments, Memory Deficits, and Neuropathology in Aged Tau Transgenic Mice Are Dependent on Leukotrienes Biosynthesis: Role of the cdk5 Kinase Pathway.

Previous studies showed that the leukotrienes pathway is increased in human tauopathy and that its manipulation may modulate the onset and development of the pathological phenotype of tau transgenic mice. However, whether interfering with leukotrienes biosynthesis is beneficial after the behavioral deficits and the neuropathology have fully developed in these mice is not known. To test this hypothesis, aged tau transgenic mice were randomized to receive zileuton, a specific leukotriene biosynthesis inhibitor, or vehicle starting at 12months of age for 16weeks and then assessed in their functional and pathological phenotype. Compared with baseline, we observed that untreated tau mice had a worsening of their memory and spatial learning. By contrast, tau mice treated with zileuton had a reversal of these deficits and behaved in an undistinguishable manner from wild-type mice. Leukotriene-inhibited tau mice had an amelioration of synaptic integrity, lower levels of neuroinflammation, and a significant reduction in tau phosphorylation and pathology, which was secondary to an involvement of the cdk5 kinase pathway. Taken together, our findings represent the first demonstration that the leukotriene biosynthesis is functionally involved at the later stages of the tau pathological phenotype and represents an ideal target with viable therapeutic potential for treating human tauopathies.

Leukotriene receptors as potential therapeutic targets.

Leukotrienes, a class of arachidonic acid-derived bioactive molecules, are known as mediators of allergic and inflammatory reactions and considered to be important drug targets. Although an inhibitor of leukotriene biosynthesis and antagonists of the cysteinyl leukotriene receptor are clinically used for bronchial asthma and allergic rhinitis, these medications were developed before the molecular identification of leukotriene receptors. Numerous studies using cloned leukotriene receptors and genetically engineered mice have unveiled new pathophysiological roles for leukotrienes. This Review covers the recent findings on leukotriene receptors to revisit them as new drug targets.

Comparison of Effect of Leukotriene Biosynthesis Blockers and Inhibitors of Phosphodiesterase Enzyme in Patients with Bronchial Hyperreactivity.

AIM:Blocking effect of leukotriene biosynthesis–zileuton and blocking the effect of phosphodiesterase enzyme–diprophylline in the treatment of patients with bronchial asthma and bronchial increased reactivity, and tiotropium bromide as an antagonist of the muscarinic receptor studied in this work.METHODS:Parameters of the lung function are determined with Body plethysmography. The resistance of the airways (Raw) was registered and measured was intrathoracic gas volume (ITGV), and specific resistance (SRaw) was also calculated. For the research, administered was zileuton (tabl. 600 mg) and diprophylline (tabl. 150 mg).RESULTS:Two days after in-house administration of leukotriene biosynthesis blocker–zileuton (4 x 600 mg orally), on the day 3 initial values of patients measured and afterwards administered 1 tablet of zileuton, and again measured was Raw and ITGV, after 60, 90 and 120 min. and calculated was SRaw; (p < 0.01). Diprophylline administered 7 days at home in a dose of (2 x 150 mg orally), on the day 8 to same patients administered 1 tablet of diprophylline, and performed measurements of Raw, ITGV, after 60, 90 and 120 min, and calculated the SRaw (p < 0.05). Treatment of the control group with tiotropium bromide - antagonist of the muscarinic receptor (2 inh. x 0.18 mcg), is effective in removal of the increased bronchomotor tonus, by also causing the significant decrease of the resistance (Raw), respectively of the specific resistance (SRaw), (p < 0.05).CONCLUSION:Effect of zileuton in blocking of leukotriene biosynthesis is not immediate after oral administration, but the effect seen on the third day of cys-LTs’ inhibition, and leukotriene B4 (LTB4) and A4 (LTA4) in patients with bronchial reactivity and bronchial asthma, which is expressed with a high significance, (p < 0.01). Blockage of phosphodiesterase enzyme–diprophylline decreases the bronchial reactivity, which is expressed with a moderate significance, (p < 0.05).

Identification of multi-target inhibitors of leukotriene and prostaglandin E2 biosynthesis by structural tuning of the FLAP inhibitor BRP-7

Leukotrienes (LTs) and prostaglandin (PG)E2 are enzymatically produced from arachidonic acid and represent highly bioactive lipid mediators with pro-inflammatory functions. Here, we report on novel multi-target inhibitors that potently and dually interfere with 5-lipoxygenase-activating protein (FLAP) and microsomal prostaglandin E2 synthase (mPGES)-1 in LT and PGE2 biosynthesis, based on the previously identified selective FLAP inhibitor BRP-7 (8, IC50 = 0.31 μM). C (5)-substitution of the benzimidazole ring of BRP-7 by carboxylic acid and its bioisosteres provided compounds, exemplified by 57 that potently suppress LT formation (IC50 = 0.05 μM) by targeting FLAP along with inhibition of mPGES-1 (IC50 = 0.42 μM). Besides FLAP, also 5-lipoxygenase (5-LO) and LTC4 synthase activities were inhibited by 57, albeit with lower potency (IC50 = 0.6 and 6.2 μM) than FLAP. Docking studies and molecular dynamic simulations with FLAP, mPGES-1 and 5-LO provide valuable insights into potential binding interactions of the inhibitors with their targets. Together, these novel benzimidazole derivatives may possess potential as leads for development of effective anti-inflammatory drugs with multi-target properties for dually inhibiting LT and PGE2 production.

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 5α-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.

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.

Synthesis and biological evaluation of C(5)-substituted derivatives of leukotriene biosynthesis inhibitor BRP-7

Pharmacological intervention with 5-lipoxygenase (5-LO) pathway leading to suppression of leukotriene (LT) biosynthesis is a clinically validated strategy for treatment of respiratory and cardiovascular diseases such as asthma and atherosclerosis. Here we describe the synthesis of a series of C(5)-substituted analogues of the previously described 5-LO-activating protein (FLAP) inhibitor BRP-7 (IC50 = 0.31 μM) to explore the effects of substitution at the C(5)-benzimidazole (BI) ring as a strategy to increase the potency against FLAP-mediated 5-LO product formation. Incorporation of polar substituents on the C(5) position of the BI core, exemplified by compound 11 with a C(5)-nitrile substituent, significantly enhances the potency for suppression of 5-LO product synthesis in human neutrophils (IC50 = 0.07 μM) and monocytes (IC50 = 0.026 μM).

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.

The Intracellular Localisation and Phosphorylation Profile of the Human 5-Lipoxygenase Δ13 Isoform Differs from That of Its Full Length Counterpart.

5-Lipoxygenase (5-LO) catalyzes leukotriene (LT) biosynthesis by a mechanism that involves interactions with 5-lipoxygenase activating protein (FLAP) and coactosin-like protein (CLP). 5-LO splice variants were recently identified in human myeloid and lymphoid cells, including the catalytically inactive ∆13 isoform (5-LO∆13) whose transcript lacks exon 13. 5-LO∆13 inhibits 5-LO product biosynthesis when co-expressed with active full length 5-LO (5-LO1). The objective of this study was to investigate potential mechanisms by which 5-LO∆13 interferes with 5-LO product biosynthesis in transfected HEK293 cells. When co-expressed with 5-LO1, 5-LO∆13 inhibited LT but not 5-hydroxyeicosatetraenoic acid (5-HETE) biosynthesis. This inhibition was independent of 5-LO∆13—FLAP interactions since it occurred in cells expressing FLAP or not. In cell-free assays CLP enhances 5-LO activity through interactions with tryptophan-102 of 5-LO. In the current study, the requirement for W102 was extended to whole cells, as cells expressing the 5-LO1-W102A mutant produced little 5-LO products. W102A mutants of 5-LO∆13 inhibited 5-LO product biosynthesis as effectively as 5-LO∆13 suggesting that inhibition is independent of interactions with CLP. Confocal microscopy showed that 5-LO1 was primarily in the nucleoplasm whereas W102A mutants showed a diffuse cellular expression. Despite the retention of known nuclear localisation sequences, 5-LO∆13 was cytosolic and concentrated in ER-rich perinuclear regions where its effect on LT biosynthesis may occur. W102A mutants of 5-LO∆13 showed the same pattern. Consistent with subcellular distribution patterns, 5-LO∆13 was hyper-phosphorylated on S523 and S273 compared to 5-LO1. Together, these results reveal a role for W102 in nuclear targeting of 5-LO1 suggesting that interactions with CLP are required for nuclear localization of 5-LO1, and are an initial characterisation of the 5-LO∆13 isoform whose inhibition of LT biosynthesis appears independent of interactions with CLP and FLAP. Better knowledge of the regulation and properties of alternative 5-LO isoforms will contribute to understanding the complex regulation of LT biosynthesis.

MK591, a second generation leukotriene biosynthesis inhibitor, prevents invasion and induces apoptosis in the bone-invading C4-2B human prostate cancer cells: implications for the treatment of castration-resistant, bone-metastatic prostate cancer.

Castration-resistant prostate cancer (CRPC) is a major clinical challenge for which no cure is currently available primarily because of the lack of proper understanding about appropriate molecular target(s). Previously we observed that inhibition of 5-lipoxygenase (5-Lox) activity induces apoptosis in some types of prostate cancer cells, suggesting an important role of 5-Lox in the viability of prostate cancer cells. However, nothing is known about the role of 5-Lox in the survival of castration-resistant, metastatic prostate cancer cells. Thus, we tested the effects of MK591, a second-generation, specific inhibitor of 5-Lox activity, on the viability and metastatic characteristics of CRPC cells. We observed that MK591 effectively kills the bone-invading C4-2B human prostate cancer cells (which bear characteristics of CRPC), but does not affect normal, non-cancer fibroblasts (which do not express 5-Lox) in the same experimental conditions. We also observed that MK591 dramatically inhibits the in vitro invasion and soft-agar colony formation of C4-2B cells. Interestingly, we found that treatment with MK591 dramatically down-regulates the expression of c-Myc and its targets at sub-lethal doses. In light of frequent over-activation of c-Myc in a spectrum of aggressive cancers (including CRPC), and the challenges associated with inhibition of c-Myc (because of its non-enzymatic nature), our novel findings of selective killing, and blockade of invasive and soft-agar colony-forming abilities of the castration-resistant, bone-metastatic C4-2B prostate cancer cells by MK591, open up a new avenue to attack CRPC cells for better management of advanced prostate cancer while sparing normal, non-cancer body cells.

5-Lipoxygenase activating protein (FLAP) dependent leukotriene biosynthesis inhibition (MK591) attenuates Lipid A endotoxin-induced inflammation.

The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.

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 (R2=0.999) and 0.26μM for the (S)-enantiomer (R2=0.986).

Inhibitory activity of IAA and IBA against lipoxygenase: in silico and in vitro validation

Inhibition of leukotriene biosynthesis is considered to be one of the potential treatment strategies for controlling inflammation, respiratory diseases and many neurodegenerative disorders. Designing of specific functional inhibitors against Lipoxygenases (LOX) has got considerable attention due to its ability to block leukotriene biosynthesis. Molecular docking analysis of two indole derivatives such as indoleacetic acid (IAA) and indolebutyric acid (IBA) are reported here. Both compounds give glide scores better than that of protocatechuic acid and nitro catechol, the two known LOX inhibitors. From the enzyme kinetic analysis, it was revealed that IAA and IBA inhibit competitively. The IC50 values determined for both IAA and IBA were 42.98 μM and 17.82 μM, respectively. The binding free energy of these compounds was determined using isothermal titration calorimetric assay and was found to be − 6.12 kcal/mol for IAA and − 7.84 kcal/mol for IBA. From the analysis, it can be concluded that both IAA and IBA might be useful as anti-inflammatory agents.