Carlo Pergola

ERK-mediated regulation of leukotriene biosynthesis by androgens: A molecular basis for gender differences in inflammation and asthma

5-Lipoxygenase initiates the biosynthesis of leukotrienes, lipid mediators involved in normal host defense and in inflammatory and allergic disorders. Despite an obvious gender bias in leukotriene-related diseases (e.g., asthma), gender aspects have been neglected in studies on leukotrienes and 5-lipoxygenase. Here, we show that leukotriene formation in stimulated whole blood or neutrophils from males is substantially lower compared with females, accompanied by changed 5-lipoxygenase trafficking. This is due to gender-specific differential activation of extracellular signal-regulated kinases (ERKs). The differences are directly related to variant male/female testosterone plus 5α-dihydrotestosterone levels, and addition of 5α-dihydrotestosterone to female blood or neutrophils reduced the high (female) LT biosynthesis capacity to low (male) levels. In conclusion, regulation of ERKs and leukotriene formation by androgens constitutes a molecular basis for gender differences in the inflammatory response, and in inflammatory diseases such as asthma.

5-Lipoxygenase inhibitors: a review of recent developments and patents

Importance to the field: Leukotrienes (LTs) are pivotal lipid mediators of inflammation and allergy and results from recent studies also suggest roles of LTs in cardiovascular diseases, cancer and osteoporosis. 5-Lipoxygenase (5-LO) catalyzes the first step in the biosynthesis of LTs from arachidonic acid, and based on the multiple potent pathophysiological actions of LTs, the pharmacological intervention with 5-LO is a challenge in the development of therapeutics. Areas covered in this review: We first summarize the biochemical regulation of 5-LO and the general pharmacological concepts in 5-LO inhibition by currently available compounds, and subsequently we report recent developments deduced from recent scientific publications and patents. What the reader will gain: A comprehensive overview about the different molecular pharmacological strategies to inhibit 5-LO and the most successful previous 5-LO inhibitors. The review also gives insights into novel concepts, for example, dual prostaglandin/LT synthesis inhibition and reveals novel compounds patented or developed within the past 5 years. Take home message: Despite the increasing therapeutic indications of anti-LT therapy, the progress in the development of novel 5-LO inhibitors is moderate. However, novel molecular concepts in the intervention with LT biosynthesis seem promising.

Myrtucommulone from Myrtus communis exhibits potent anti-inflammatory effectiveness in vivo.

Myrtucommulone (MC), a nonprenylated acylphloroglucinol contained in the leaves of myrtle (Myrtus communis), has been reported to suppress the biosynthesis of eicosanoids by inhibition of 5-lipoxygenase and cyclooxygenase-1 in vitro and to inhibit the release of elastase and the formation of reactive oxygen species in activated polymorphonuclear leukocytes. Here, in view of the ability of MC to suppress typical proinflammatory cellular responses in vitro, we have investigated the effects of MC in in vivo models of inflammation. MC was administered to mice intraperitoneally, and paw edema and pleurisy were induced by the subplantar and intrapleural injection of carrageenan, respectively. MC (0.5, 1.5, and 4.5 mg/kg i.p.) reduced the development of mouse carrageenan-induced paw edema in a dose-dependent manner. Moreover, MC (4.5 mg/kg i.p. 30 min before and after carrageenan) exerted anti-inflammatory effects in the pleurisy model. In particular, 4 h after carrageenan injection in the pleurisy model, MC reduced: 1) the exudate volume and leukocyte numbers; 2) lung injury (histological analysis) and neutrophil infiltration (myeloperoxidase activity); 3) the lung intercellular adhesion molecule-1 and P-selectin immunohistochemical localization; 4) the cytokine levels (tumor necrosis factor-α and interleukin-1β) in the pleural exudate and their immunohistochemical localization in the lung; 5) the leukotriene B4, but not prostaglandin E2, levels in the pleural exudates; and 6) lung peroxidation (thiobarbituric acid-reactant substance) and nitrotyrosine and poly (ADP-ribose) immunostaining. In conclusion, our results demonstrate that MC exerts potent anti-inflammatory effects in vivo and offer a novel therapeutic approach for the management of acute inflammation.

Structural Optimization and Biological Evaluation of 2-Substituted 5-Hydroxyindole-3-carboxylates as Potent Inhibitors of Human 5-Lipoxygenase

Pharmacological suppression of leukotriene biosynthesis by inhibitors of 5-lipoxygenase (5-LO) is a strategy to intervene with inflammatory and allergic disorders. We recently presented 2-amino-5-hydroxy-1H-indoles as efficient 5-LO inhibitors in cell-based and cell-free assays. Structural optimization led to novel benzo[g]indole-3-carboxylates exemplified by ethyl 2-(3-chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (compound 11a), which inhibits 5-LO activity in human neutrophils and recombinant human 5-LO with IC(50) values of 0.23 and 0.086 microM, respectively. Notably, 11a efficiently blocks 5-LO product formation in human whole blood assays (IC(50) = 0.83-1.6 microM) and significantly prevented leukotriene B(4) production in pleural exudates of carrageenan-treated rats, associated with reduced severity of pleurisy. Together, on the basis of their high potency against 5-LO and the marked efficacy in biological systems, these novel and straightforward benzo[g]indole-3-carboxylates may have potential as anti-inflammatory therapeutics.

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

The molecular mechanism of the inhibition by licofelone of the biosynthesis of 5-lipoxygenase products.

Licofelone is a dual inhibitor of the cyclooxygenase and 5‐lipoxygenase (5‐LO) pathway, and has been developed for the treatment of inflammatory diseases. Here, we investigated the molecular mechanisms underlying the inhibition by licofelone of the formation of 5‐LO products.

The Molecular Pharmacology and In Vivo Activity of 2-(4-Chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid (YS121), a Dual Inhibitor of Microsomal Prostaglandin E2 Synthase-1 and 5-Lipoxygenase

The microsomal prostaglandin E2 synthase (mPGES)-1 is one of the terminal isoenzymes of prostaglandin (PG) E2 biosynthesis. Pharmacological inhibitors of mPGES-1 are proposed as an alternative to nonsteroidal anti-inflammatory drugs. We recently presented the design and synthesis of a series of pirinixic acid derivatives that dually inhibit mPGES-1 and 5-lipoxygenase. Here, we investigated the mechanism of mPGES-1 inhibition, the selectivity profile, and the in vivo activity of α-(n-hexyl)-substituted pirinixic acid [YS121; 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)octanoic acid)] as a lead compound. In cell-free assays, YS121 inhibited human mPGES-1 in a reversible and noncompetitive manner (IC50 = 3.4 μM), and surface plasmon resonance spectroscopy studies using purified in vitro-translated human mPGES-1 indicate direct, reversible, and specific binding to mPGES-1 (KD = 10–14 μM). In lipopolysaccharide-stimulated human whole blood, PGE2 formation was concentration dependently inhibited (IC50 = 2 μM), whereas concomitant generation of the cyclooxygenase (COX)-2-derived thromboxane B2 and 6-keto PGF1α and the COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was not significantly reduced. In carrageenan-induced rat pleurisy, YS121 (1.5 mg/kg i.p.) blocked exudate formation and leukocyte infiltration accompanied by reduced pleural levels of PGE2 and leukotriene B4 but also of 6-keto PGF1α. Taken together, these results indicate that YS121 is a promising inhibitor of mPGES-1 with anti-inflammatory efficiency in human whole blood as well as in vivo.

Discovery of benzo[g]indol-3-carboxylates as potent inhibitors of microsomal prostaglandin E2 synthase-1

Selective inhibition of pro-inflammatory prostaglandin (PG)E(2) formation via microsomal PGE(2) synthase-1 (mPGES-1) might be superior over inhibition of all cyclooxygenase (COX)-derived products by non-steroidal anti-inflammatory drugs (NSAIDs) and coxibs. We recently showed that benzo[g]indol-3-carboxylates potently suppress leukotriene biosynthesis by inhibiting 5-lipoxygenase. Here, we describe the discovery of benzo[g]indol-3-carboxylates as a novel class of potent mPGES-1 inhibitors (IC(50)0.1 microM). Ethyl 2-(3-chlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (compound 7a) inhibits human mPGES-1 in a cell-free assay (IC(50)=0.6 microM) as well as in intact A549 cells (IC(50)=2 microM), and suppressed PGE(2) pleural levels in rat carrageenan-induced pleurisy. Inhibition of cellular COX-1/2 activity was significantly less pronounced. Compound 7a significantly reduced inflammatory reactions in the carrageenan-induced mouse paw edema and rat pleurisy. Together, based on the select and potent inhibition of mPGES-1 and 5-lipoxygenase, benzo[g]indol-3-carboxylates possess potential as novel anti-inflammatory drugs with a valuable pharmacological profile.

Synthesis, biological evaluation, and enzyme docking simulations of 1,5-diarylpyrrole-3-alkoxyethyl ethers as selective cyclooxygenase-2 inhibitors endowed with anti-inflammatory and antinociceptive activity.

A series of substituted 1,5-diarylpyrrole-3-alkoxyethyl ethers (6, 7, and 8) has been synthesized with the aim to assess if in the previously reported 1,5-diarylpyrrole derivatives (5) the replacement of the acetic ester moiety with an alkoxyethyl group still led to new, highly selective and potent COX-2 inhibitors. In the in vitro cell culture assay, all the compounds proved to be potent and selective COX-2 inhibitors. In the human whole blood (HWB) assay, compound 8a had a comparable COX-2 selectivity to valdecoxib, while it was more selective than celecoxib but less selective than rofecoxib. The potential anti-inflammatory and antinociceptive activities of compounds 7a, 8a, and 8d were evaluated in vivo, where they showed a very good activity against both carrageenan-induced hyperalgesia and edema in the rat paw test. In the abdominal constriction test compound 7a, 8a, and 8d were able to reduce the number of writhes in a statistically significant manner. Furthermore, the affinity data of these compounds have been rationalized through enzyme docking simulations in terms of interactions with a crystallographic model of the COX-2 binding site by means of the software package Autodock 3.0.5, GRID 21, and MacroModel 8.5 using the complex between COX-2 and SC-558 (1b), refined at a 3 A resolution (Brookhaven Protein Data Bank entry: 6cox ).

Novel Ester and Acid Derivatives of the 1,5-Diarylpyrrole Scaffold as Anti-Inflammatory and Analgesic Agents. Synthesis and in Vitro and in Vivo Biological Evaluation

A new generation of selective cyclooxygenase-2 (COX-2) inhibitors (coxibs) was developed to circumvent the major side effects of cyclooxygenase-1 (COX-1) and COX-2 inhibitors (stomach ulceration and nephrotoxicity). As a consequence, coxibs are extremely valuable in treating acute and chronic inflammatory conditions. However, the use of coxibs, such as rofecoxib (Vioxx), was discontinued because of the high risk of cardiovascular adverse events. More recent clinical findings highlighted how the cardiovascular toxicity of coxibs could be mitigated by an appropriate COX-1 versus COX-2 selectivity. We previously reported a set of substituted 1,5-diarylpyrrole derivatives, selective for COX-2. Here, we describe the synthesis of new 1,5-diarylpyrroles along with their inhibitory effects in vitro, ex vivo, and in vivo toward COX isoenzymes and their analgesic activity. Isopropyl-2-methyl-5-[4-(methylsulfonyl)phenyl]-1-phenyl-1H-pyrrole-3-acetate (10a), a representative member of the series, was selected for pharmacokinetic and metabolic studies.