Identification of new γ-hydroxybutenolides that preferentially inhibit the activity of mPGES-1

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) has been recognized as novel, promising drug target for anti-inflammatory and anticancer drugs. mPGES-1 catalyzes the synthesis of the inducible prostaglandin E(2) in response to pro-inflammatory stimuli, rendering this enzyme extremely interesting in drug discovery process owing to the drastic reduction of the severe side effects typical for traditional non-steroidal anti-inflammatory drugs. In the course of our investigations focused on this topic, we identified two interesting molecules bearing the γ-hydroxybutenolide scaffold which potently inhibit the activity of mPGES-1. Notably, the lead compound 2c that inhibited mPGES-1 with IC(50)=0.9μM, did not affect other related enzymes within the arachidonic acid cascade.     

Further insights on the structural aspects of PLA(2) inhibition by gamma-hydroxybutenolide-containing natural products: a comparative study on petrosaspongiolides M-R

Petrosaspongiolides M-R (PM-PR, 1-5) are marine sesterterpenes structurally characterised by a gamma-hydroxybutenolide moiety. They have shown an in vitro and in vivo potent anti-inflammatory activity, mediated by specific inhibition of secretory phospholipase A(2) (sPLA(2) enzymes). The molecular mechanism underlying the sub-micromolar irreversible inhibition of the bee-venom PLA(2) (bvPLA(2)) by PM has been clarified combining mass spectrometry (MS) and molecular modelling approaches. The N-terminal amino group (Ile-1 residue), recently identified as the unique PM covalent binding site on this enzyme, selectively delivers a nucleophilic attack onto the masked aldehyde at C-25 of the pharmacophoric gamma-hydroxybutenolide ring of PM, giving rise to a Schiff base. In the attempt of broadening the knowledge of the mechanism at molecular level of PLA(2) inactivation by this family of compounds, we performed a comparative analysis on petrosaspongiolides M-R, whose results are discussed in this paper. Firstly, the amount of bvPLA(2) enzyme covalently modified after incubation with each of petrosaspongiolides M-R was measured and resulted to be in good agreement with pharmacological in vitro data. Then, a full characterisation of the bvPLA(2) adduct with PR, one of the least active and most structurally different among petrosaspongiolides, by LC-MS, MS(n), and computational methods, confirmed the same inhibition mechanism and covalent binding site already found for PM. Finally, extensive molecular docking studies performed in comparison on the PM-PLA(2) and PR-PLA(2) complexes provided critical insight on how the balance between non-covalent and covalent inhibitor-enzyme interactions may affect the final potency exhibited by the various compounds of the petrosaspongiolide family.     

Biarylimidazoles as inhibitors of microsomal prostaglandin E2 synthase-1

Microsomal prostaglandin E(2) synthase (mPGES-1) represents a potential target for novel analgesic and anti-inflammatory agents. High-throughput screening identified several leads of mPGES-1 inhibitors which were further optimized for potency and selectivity. A series of inhibitors bearing a biaryl imidazole scaffold exhibits excellent inhibition of PGE(2) production in enzymatic and cell-based assays. The synthesis of these molecules and their activities will be discussed.     

Synthesis and pharmacological characterization of benzenesulfonamides as dual species inhibitors of human and murine mPGES-1

The microsomal prostaglandin E₂ synthase 1 (mPGES-1) became a desirable target in recent years for the research of new anti-inflammatory drugs. Even though many potent inhibitors of human mPGES-1, tested in vitro assay systems, have been synthesized, they all failed in preclinical trials in rodent models of inflammation, due to the lack of activity on rodent enzyme. Within this work we want to present a new class of mPGES-1 inhibitors derived from a benzenesulfonamide scaffold with inhibitory potency on human and murine mPGES-1. Starting point with an IC₅₀ of 13.8 μM on human mPGES-1 was compound 1 (4-{benzyl[(4-methoxyphenyl)methyl]sulfamoyl}benzoic acid; FR4), which was discovered by a virtual screening approach. Optimization during a structure–activity relationship (SAR) process leads to compound 28 (4-[(cyclohexylmethyl)[(4-phenylphenyl)methyl]sulfamoyl]benzoic acid) with an improved IC₅₀ of 0.8 μM on human mPGES-1. For the most promising compounds a broad pharmacological characterization has been carried out to estimate their anti-inflammatory potential.     

Signal pathways involved in the regulation of prostaglandin E synthase-1 in human gingival fibroblasts

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme regulating the synthesis of prostaglandin E2 (PGE2) in inflammatory conditions. In this study we investigated the regulation of mPGES-1 in gingival fibroblasts stimulated with the inflammatory mediators interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNFalpha). The results showed that IL-1beta and TNFalpha induce the expression of mPGES-1 without inducing the expression of early growth response factor-1 (Egr-1). Treatment of the cells with the PLA2 inhibitor 4-bromophenacyl bromide (BPB) decreased the cytokine-induced mPGES-1 expression accompanied by decreased PGE2 production whereas the addition of arachidonic acid (AA) upregulated mPGES-1 expression and PGE2 production. The protein kinase C (PKC) activator PMA did not upregulate the expression of mPGES-1 in contrast to COX-2 expression and PGE2 production. In addition, inhibitors of PKC, tyrosine and p38 MAP kinase markedly decreased the cytokine-induced PGE2 production but not mPGES-1 expression. Moreover, the prostaglandin metabolites PGE2 and PGF2alpha induced mPGES-1 expression as well as upregulated the cytokine-induced mPGES-1 expression indicating positive feedback regulation of mPGES-1 by prostaglandin metabolites. The peroxisome proliferator-activated receptor-gamma (PPARgamma) ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), decreased mPGES-1 expression but not COX-2 expression or PGE2 production. The results indicate that the inflammatory-induced mPGES-1 expression is regulated by PLA2 and 15d-PGJ2 but not by PKC, tyrosine kinase or p38 MAP kinase providing new insights into the regulation of mPGES-1.     

Synthesis and biological activity of cyclotetrapeptide analogues of the natural HDAC inhibitor FR235222

In the course of our ongoing efforts to discover new and more effective HDAC inhibitors useful for the development of promising anticancer candidates, we have recently undertaken a molecular modelling study on a small collection of FR235222 analogues, synthesized by us in the frame of a structure–activity relationship investigation, made in order to identify the key structural elements essential for the activity. Progress made in structure elucidation of HDAC active site, together with accurate docking calculations, provided new structural insights useful for a further refinement of the tetrapeptide scaffold which should assure an optimal interaction between the synthetic ligands and the biological target. Following the computer aided suggestions we synthesized six new cyclotetrapeptide analogues of the lead compound (3–8), bearing a carboxylic or an hydroxamic acid functionality as Zn binding moiety. Herein we describe their synthesis and their inhibition activity on different HDAC isoforms.     

Fragment-based discovery of novel and selective mPGES-1 inhibitors Part 1: Identification of sulfonamido-1,2,3-triazole-4,5-dicarboxylic acid

Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible prostaglandin E synthase that catalyzes the conversion of prostaglandin PGH₂ to PGE₂ and represents a novel target for therapeutic treatment of inflammatory disorders. It is essential to identify mPGES-1 inhibitor with novel scaffold as new hit or lead compound for the purpose of the next-generation anti-inflammatory drugs. Herein we report the discovery of sulfonamido-1,2,3-triazole-4,5-dicarboxylic derivatives as a novel class of mPGES-1 inhibitors identified through fragment-based virtual screening and in vitro assays on the inhibitory activity of the actual compounds. 1-[2-(N-Phenylbenzenesulfonamido)ethyl]-1H-1,2,3-triazole-4,5-dicarboxylic acid (6f) inhibits human mPGES-1 (IC₅₀ of 1.1 μM) with high selectivity (ca.1000-fold) over both COX-1 and COX-2 in a cell-free assay. In addition, the activity of compound 6f was again tested at 10 μM concentration in presence of 0.1% Triton X-100 and found to be reduced to 1/4 of its original activity without this detergent. Compared to the complete loss of activity of nuisance inhibitor with the detergent, therefore, compound 6f would be regarded as a partial nuisance inhibitor of mPGES-1 with a novel scaffold for the optimal design of more potent mPGES-1 inhibitors.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits the expression of microsomal prostaglandin E synthase type 2 in colon cancer cells

Prostaglandin (PG) E(2) (PGE(2)) plays a predominant role in promoting colorectal carcinogenesis. The biosynthesis of PGE(2) is accomplished by conversion of the cyclooxygenase (COX) product PGH(2) by several terminal prostaglandin E synthases (PGES). Among the known PGES isoforms, microsomal PGES type 1 (mPGES-1) and type 2 (mPGES-2) were found to be overexpressed in colorectal cancer (CRC); however, the role and regulation of these enzymes in this malignancy are not yet fully understood. Here, we report that the cyclopentenone prostaglandins (CyPGs) 15-deoxy-Delta(12,14)-PGJ(2) and PGA(2) downregulate mPGES-2 expression in the colorectal carcinoma cell lines Caco-2 and HCT 116 without affecting the expression of any other PGES or COX. Inhibition of mPGES-2 was subsequently followed by decreased microsomal PGES activity. These effects were mediated via modulation of the cellular thiol-disulfide redox status but did not involve activation of the peroxisome proliferator-activated receptor gamma or PGD(2) receptors. CyPGs had antiproliferative properties in vitro; however, this biological activity could not be directly attributed to decreased PGES activity because it could not be reversed by adding PGE(2). Our data suggest that there is a feedback mechanism between PGE(2) and CyPGs that implicates mPGES-2 as a new potential target for pharmacological intervention in CRC.     

核苷类药物酶法合成研究进展

由于核苷类似物具有很高的抗病毒活性,因为而已成为医药工作者研究的重点。运用酶法合成核苷类似物．已经显示了巨大的优势。本文综述了酶法合成核苷类似物的产生菌种和酶系,以及它们的催化机理,并罗列了已经用于生产或较有使用价值的菌种。     

Improved gelatinase a selectivity by novel zinc binding groups containing galardin derivatives

The synthesis of several analogues of galardin, a MMP inhibitor, are presented with their in vitro inhibitory activity against MMP-1 and MMP-2. These compounds contain a distinct Zinc Binding Group (ZBG). Those having a 2-acylated-heterocycle as well as a 2-arylamide function do not exhibit a good inhibition/selectivity against the enzymes tested. On the contrary, those that are based on a hydrazide scaffold present potent selectivity for MMP-2 versus MMP-1.     

Petrosaspongiolide M reduces morphine withdrawal in vitro

The bee venom phospholipase A(2) (PLA(2)) inhibitory activity of petrosaspongiolide M (PM), a marine metabolite displaying a potent anti-inflammatory activity and able to covalently bind and block group II and III secretory PLA(2) enzymes, has been investigated by mass spectrometry and molecular modeling. The model reveals interesting insight on the PM-PLA(2) inhibition process and may prove useful in the design of new anti-inflammatory agents targeting PLA(2) secretory enzymes. In this paper, the effect of PM has been investigated on opiate withdrawal in an in vitro model. After a 4 min in vitro exposure to morphine a strong contracture of guinea pig isolated ileum was observed after the addition of naloxone. PM treatment 1 x 10(-8), 5 x 10(-8), 1 x 10(-7) M was able to reduce morphine withdrawal. These results suggest that PM effect in this in vitro model of opiate withdrawal may be due to extracellular type II PLA(2) inhibition.     

Cyclooxygenase-1 and -2 enzymes differentially regulate the brain upstream NF-kappa B pathway and downstream enzymes involved in prostaglandin biosynthesis

We have recently reported that cyclooxygenase (COX)-2-deficiency affects brain upstream and downstream enzymes in the arachidonic acid (AA) metabolic pathway to prostaglandin E2 (PGE2), as well as enzyme activity, protein and mRNA levels of the reciprocal isozyme, COX-1. To gain a better insight into the specific roles of COX isoforms and characterize the interactions between upstream and downstream enzymes in brain AA cascade, we examined the expression and activity of COX-2 and phospholipase A2 enzymes (cPLA2 and sPLA2), as well as the expression of terminal prostaglandin E synthases (cPGES, mPGES-1, and - 2) in wild type and COX-1(-/-) mice. We found that brain PGE2 concentration was significantly increased, whereas thromboxane B2 (TXB2) concentration was decreased in COX-1(-/-) mice. There was a compensatory up-regulation of COX-2, accompanied by the activation of the NF-kappaB pathway, and also an increase in the upstream cPLA2 and sPLA2 enzymes. The mechanism of NF-kappaB activation in the COX-1(-/-) mice involved the up-regulation of protein expression of the p50 and p65 subunits of NF-kappaB, as well as the increased protein levels of phosphorylated IkappaBalpha and of phosphorylated IKKalpha/beta. Overall, our data suggest that COX-1 and COX-2 play a distinct role in brain PG biosynthesis, with basal PGE2 production being metabolically coupled with COX-2 and TXB2 production being preferentially linked to COX-1. Additionally, COX-1 deficiency can affect the expression of reciprocal and coupled enzymes, COX-2, Ca2+ -dependent PLA2, and terminal mPGES-2, to overcome defects in brain AA cascade.     

Development of 2-aryl substituted quinazolin-4(3H)-one,pyrido[4,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4(3H)-one derivatives as microsomal prostaglandin E2 synthase-1 inhibitors

mPGES-1 is inducible terminal synthase acting downstream of COX enzymes in arachidonic acid pathway, regulates the biosynthesis of pro-inflammatory prostaglandin PGE₂. Cardiovascular side effect of coxibs and NSAIDs, selective for COX-2 inhibition, stimulated interest in mPGES-1, a therapeutic target with potential to deliver safe and effective anti-inflammatory drugs. The synthesis and structure activity relationship of a series of compounds from 2-aryl substituted quinazolin-4(3H)-one, pyrido[4,3-d]pyrimidin-4(3H)-one and pyrido[2,3-d]pyrimidin-4(3H)-one scaffolds as mPGES-1 inhibitor are discussed. A set of analogs (28, 48, 49) were identified with <10 nM potencies in the recombinant human mPGES-1 enzyme and in the A549 cellular assays. These analogs were also found to be potent in the human whole blood assay (<400 nM). Furthermore, the representative compound 48 was shown to be selective with other prostanoid synthases and was able to effectively regulate PGE₂ biosynthesis in clinically relevant inflammatory settings, in comparison with celecoxib.     

Design of new potent and selective secretory phospholipase A2 inhibitors. Part 5: synthesis and biological activity of 1-alkyl-4-[4,5-dihydro-1,2,4-[4H]-oxadiazol-5-one-3-ylmethylbenz-4'-yl(oyl)] piperazines

Among the different PLA(2)s identified to date, the group IIA secretory PLA(2) (sPLA(2) GIIA) is implied in diverse pathological conditions. In this work we describe the synthesis, inhibitory activities, and structure-activity relationships (SAR) of a new class of substituted piperazine derivatives. The in vitro fluorimetric assay using two groups of enzymes, GIB and GIIA, revealed several compounds as highly potent inhibitors (IC(50)=0.1 microM). The in vivo activity assessed by ip or per os administration in a carrageenan-induced edema test in rats showed that two compounds proved to be as potent as indomethacin (10 mg/kg).     

Phospholipase A(2) regulation of arachidonic acid mobilization

Phospholipase A(2) (PLA(2)) constitutes a growing superfamily of lipolytic enzymes, and to date, at least 19 distinct enzymes have been found in mammals. This class of enzymes has attracted considerable interest as a pharmacological target in view of its role in lipid signaling and its involvement in a variety of inflammatory conditions. PLA(2)s hydrolyze the sn-2 ester bond of cellular phospholipids, producing a free fatty acid and a lysophospholipid, both of which are lipid signaling molecules. The free fatty acid produced is frequently arachidonic acid (AA, 5,8,11,14-eicosatetraenoic acid), the precursor of the eicosanoid family of potent inflammatory mediators that includes prostaglandins, thromboxanes, leukotrienes and lipoxins. Multiple PLA(2) enzymes are active within and surrounding the cell and these enzymes have distinct, but interconnected roles in AA release.     

1,2,5-Thiadiazolidin-3-one 1,1-dioxide-based heterocyclic sulfides are potent inhibitors of human tryptase

We describe herein the design, synthesis, and in vitro biochemical evaluation of a series of potent, time-dependent inhibitors of the mast cell-derived serine protease tryptase. The inhibitors were readily obtained by attaching various heterocyclic thiols, as well as a basic primary specificity residue P₁, to the 1,2,5-thiadiazolidin-3-one 1,1-dioxide scaffold. The inhibitors were found to be devoid of any inhibitory activity toward a neutral (elastase) or cysteine (papain) protease, however they were also fairly efficient inhibitors of bovine trypsin. The differential inhibition observed with trypsin suggests that enzyme selectivity can be optimized by exploiting differences in the S′ subsites of the two enzymes. The results described herein demonstrate the versatility of the heterocyclic scaffold in fashioning mechanism-based inhibitors of neutral, basic, and acidic (chymo)trypsin-like serine proteases.     

Prostaglandin E synthase, a terminal enzyme for prostaglandin E2 biosynthesis

Biosynthesis of prostanoids is regulated by three sequential enzymatic steps, namely phospholipase A2 enzymes, cyclooxygenase (COX) enzymes, and various lineagespecific terminal prostanoid synthases. Prostaglandin E synthase (PGES), which isomerizes COX-derived PGH2 specifically to PGE2, occurs in multiple forms with distinct enzymatic properties, expressions, localizations and functions. Two of them are membrane-bound enzymes and have been designated as mPGES-1 and mPGES-2. mPGES-1 is a perinuclear protein that is markedly induced by proinflammatory stimuli, is down-regulated by antiinflammatory glucocorticoids, and is functionally coupled with COX-2 in marked preference to COX-1. Recent gene targeting studies of mPGES-1 have revealed that this enzyme represents a novel target for anti-inflammatory and anti-cancer drugs. mPGES-2 is synthesized as a Golgi membrane-associated protein, and the proteolytic removal of the N-terminal hydrophobic domain leads to the formation of a mature cytosolic enzyme. This enzyme is rather constitutively expressed in various cells and tissues and is functionally coupled with both COX-1 and COX-2. Cytosolic PGES (cPGES) is constitutively expressed in a wide variety of cells and is functionally linked to COX-1 to promote immediate PGE2 production. This review highlights the latest understanding of the expression, regulation and functions of these three PGES enzymes.     

Effect of D-alanylation of (lipo)teichoic acids of Staphylococcus aureus on host secretory phospholipase A2 action before and after phagocytosis by human neutrophils

Invading bacteria such as Staphylococcus aureus induce mobilization of professional phagocytes (e.g., neutrophils) and extracellular antibacterial proteins (e.g., group IIA phospholipase A2 (gIIA PLA2)). Accumulation of gIIA PLA2 in inflammatory fluids confers potent extracellular antistaphylococcal activity and at lower concentrations promotes bacterial phospholipid degradation during phagocytosis of S. aureus by human neutrophils. D-alanylation of (lipo) teichoic acids of S. aureus increases bacterial resistance to gIIA PLA2 approximately 100-fold, raising the possibility that the resistance of ingested S. aureus to related gV and gX secretory PLA2 present in human neutrophil granules depends on D-alanylation mediated by the dlt operon. However, we show that isogenic wild-type and dltA S. aureus are equally resistant to gV/X PLA2 during phagocytosis and when exposed to the purified enzymes. The fates of wild-type and dltA S. aureus exposed to serum and human neutrophils differed significantly only when extracellular gIIA PLA2 was also present before phagocytosis. The extreme potency of the gIIA PLA2 toward dltA S. aureus suggests that even small amounts of this extracellular enzyme mobilized early in inflammation could contribute substantially to the overall cytotoxicity of acute inflammatory exudates toward S. aureus when D-alanylation of (lipo)teichoic acids is limiting.