Novel di-tertiary-butyl phenylhydrazones as dual cyclooxygenase-2/5-lipoxygenase inhibitors: Synthesis,COX/LOX inhibition,molecular modeling,and insights into their cytotoxicities

Although dual inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) enzymes is highly effective than targeting COX or LOX alone, there are only a few reports of examining such compounds in case of colorectal cancers (CRC). In the present work we report that the novel di-tert-butyl phenol-based dual inhibitors DTPSAL, DTPBHZ, DTPINH, and DTPNHZ exhibit significant cytotoxicity against human CRC cell lines. Molecular docking studies revealed a good fit of these compounds in the COX-2 and 5-LOX protein cavities. The inhibitors show significant inhibition of COX-2 and 5-LOX activities and are effective against a panel of human colon cancer cell lines including HCA-7, HT-29, SW480 and intestinal Apc10.1 cells as well as the hyaluronan synthase-2 (Has2) enzyme over-expressing colon cancer cells, through inhibition of the Hyaluronan/CD44v6 cell survival pathway. Western blot analysis and qRT-PCR analyses indicated that the di-tert-butyl phenol-based dual inhibitors reduce the expression of COX-2, 5-LOX, and CD44v6 in human colon cancer HCA-7 cells, while the combination of CD44v6shRNA and DTPSAL has an additional inhibitory effect on CD44v6 mRNA expression. The synergistic inhibitory effect of Celecoxib and Licofelone on CD44v6 mRNA expression suggests that the present dual inhibitors down-regulate cyclooxygenase and lipoxygenase enzymes through CD44v6. The compounds also exhibited enhanced antiproliferative potency compared to standard dual COX/LOX inhibitor, viz. Licofelone. Importantly, the HA/CD44v6 antagonist CD44v6shRNA in combination with synthetic compounds had a sensitizing effect on the cancer cells which enhanced their antiproliferative potency, a finding which is crucial for the anti-proliferative potency of the novel synthetic di-tert-butyl phenol based dual COX–LOX inhibitors in colon cancer cells.     

Synthesis and biological evaluation of acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl pharmacophore: dual inhibitors of cyclooxygenases and lipoxygenases

A new class of regioisomeric acyclic triaryl (Z)-olefins possessing a 3,5-di-tert-butyl-4-hydroxyphenyl (DTBHP) 5-lipoxygenase (5-LOX) pharmacophore that is vicinal to a para-methanesulfonylphenyl cyclooxygenase-2 (COX-2) pharmacophore were designed for evaluation as selective COX-2 and/or 5-LOX inhibitors. The target compounds were synthesized via a highly stereoselective McMurry olefination cross-coupling reaction. This key synthetic step afforded a (Z):(E) olefinic mixture with a predominance for the (Z)-olefin stereoisomer. Structure-activity studies for the (Z)-1-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-(4-methanesulfonylphenyl)-1-phenylalk-1-ene regioisomers showed that COX-1 inhibition decreased, COX-2 inhibition increased, and the COX-2 selectivity index (SI) increased as the chain length of the alkyl substituent attached to the olefinic double bond was increased (Et-->n-butyl-->n-heptyl). In this group of compounds, inhibition of both 5-LOX and 15-LOX was dependent upon the length of the alkyl substituent with the hex-1-ene compound 9c having a n-butyl substituent exhibiting potent inhibition of both 5-LOX (IC50=0.3 microM) and 15-LOX (IC50=0.8 microM) relative to the inactive (IC50>10 microM) Et and n-heptyl analogs. Compound 9c is of particular interest since it also exhibits a dual inhibitory activity against the COX (COX-1 IC50=3.0 microM, and COX-2 IC50=0.36 microM, COX-2 SI=8.3) isozymes. A comparison of the relative inhibitory activities of the two groups of regioisomers investigated shows that the regioisomers in which the alkyl substituent is attached to the same olefinic carbon atom (C-2) as the para-methanesulfonylphenyl moiety generally exhibit a greater potency with respect to COX-2 inhibition. The 4-hydroxy substituent in the 3,5-di-tert-butyl-4-hydroxyphenyl moiety is essential for COX and LOX inhibition since 3,5-di-tert-butyl-4-acetoxyphenyl derivatives were inactive inhibitors. These structure-activity data indicate acyclic triaryl (Z)-olefins constitute a suitable template for the design of dual COX-2/LOX inhibitors.     

Modification of eicosanoid profile in human blood treated by dual COX/LOX inhibitors

The arachidonic acid metabolizing enzymes, the cyclooxygenases (COXs) and lipoxygenases (LOXs), have been implicated in the development of a variety of cancers and numerous new therapeutic inhibitors are currently under investigation. However, given the interdependence of the two pathways, the effect of inhibiting one pathway with relatively selective agents can only be appreciated in the in vivo situation. Clearly then, because of their potential beneficial or deleterious effects, it is important to understand the nature and levels of the resulting arachidonic acid metabolites when treating patients with relatively selective inhibitor drugs. In this study, using reference COX-2, 5-LOX and dual COX-2/5-LOX inhibitors, we devised a protocol which permitted the simultaneous quantification of eicosanoid metabolites formed during stimulation of human peripheral venous blood samples with the calcium ionophore, A23187, in the absence and presence of lipopolysaccharide (LPS). Not surprisingly, the end products of both COX and LOX pathways were affected depending on the inhibitor, or combination of inhibitors, used and the concentrations of drug tested. In conclusion, the method described permits the rapid screening of novel compounds for potentially positive and/or negative effects upon the products of arachidonic acid metabolism.     

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) selectivity of COX inhibitors

In vitro evaluations of the selectivity of COX inhibitors are based on a great variety of experimental protocols. As a result, data available on cyclooxygenase (COX)-1/COX-2/5- lipoxygenase (LOX) selectivity of COX inhibitors lack consistency. We, therefore, performed a systematic analysis of the COX-1/COX-2/5-LOX selectivity of 14 compounds with selective COX inhibitory activity (Coxibs). The compounds belonged to different structural classes and were analyzed employing the well-recognized whole-blood assay. 5-LOX activity was also tested on isolated human polymorphonuclear leukocytes. Among COX inhibitors, celecoxib and ML-3000 (licofelone) inhibited 5-LOX in human neutrophils at micromolar ranges. Surprisingly, ML-3000 had no effect on 5-LOX product synthesis in whole-blood assay. In addition, we could show that inhibition of COX pathways did not increase the transformation of arachidonic acid by the 5-LOX pathway.     

Enzyme selectivity of new cyclooxygenase-2/5 lipoxygenase inhibitors using molecular modeling approach

We have studied the conformational flexibility of three 5-keto-substituted 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofurans (DHDMBFs) which show dual cyclooxygenase (COX) and 5-lipoxygenase (LOX) inhibition and are potential candidates as antiinflammatory agents and analgesics. The conformations were studied by systematic search, molecular mechanics (MM) and simulated annealing molecular dynamics (SAMD) techniques. We also studied several structure based parameters and distribution of molecular electrostatic potential (MEP) around these molecules. All the three compounds were docked in the active cavity of cyclooxygenase-2 (COX-2) using graphical and energy grid search techniques. The complex geometries were optimized by MM. The results on conformational flexibility, inter-atomic distances and angles, MEP distribution and points of contacts with peptide side chains in active cavity have been used to understand the mechanistic cause of differential action of these molecules.     

Eicosanoids mediate Manduca sexta cellular response to the fungal pathogen Beauveria bassiana: a role for the lipoxygenase pathway

Many studies have documented the involvement of eicosanoids in insect cellular immune responses to bacteria. The use of the fungal pathogen Beauveria bassiana as a nodulation elicitor, with inhibition of phospholipase A(2) by dexamethasone, extends the principle to fungi. This study also provides the first evidence of involvement of the lipoxygenase (LOX) pathway rather than the cyclooxygenase (COX) pathway in synthesis of the nodulation mediating eicosanoid(s). The LOX product, 5(S)-hydroperoxyeicosa-6E,8Z,11Z,14Z-tetraenoic acid (5-HPETE), substantially reversed nodulation inhibition caused by dexamethasone and the LOX inhibitors, caffeic acid and esculetin. The COX product, prostaglandin H(2) (PGH(2)), did not reverse the nodulation inhibition by dexamethasone or the COX inhibitor, ibuprofen. None of the inhibitors tested had a significant effect on the phagocytosis of B. bassiana blastospores in vitro. Hemocyte phenoloxidase activity was reduced by dexamethasone, esculetin, and the COX inhibitor, indomethacin. The rescue candidates 5-HPETE and PGH(2) did not reverse the inhibition.     

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.     

Essential structural profile of a dual functional inhibitor against cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX): molecular docking and 3D-QSAR analyses on DHDMBF analogues

It is recently proposed that compounds with equal capabilities of inhibiting COX and 5-LOX, both are key enzymes involved in the arachidonic acid (AA) cascade, are expected to be safer non-steroidal anti-inflammatory drugs (NSAIDs). To dig out helpful information in designing dual functional inhibitors against the two enzymes, homology modeling, molecular dynamics (MD) simulations, automated docking, and 3D-QSAR analyses were performed in this study on 21 COX-2/5-LOX dual inhibitors, namely, 7-tert-butyl-2,3-dihydro-3,3-dimethylbenzofuran (DHDMBF) analogues. A 3D-model of 5-LOX was built based on the high-resolution X-ray structure of rabbit reticulocyte 15-lipoxygenase. Molecular docking was then applied to locate the binding orientations and conformations of DHDMBF analogues with COX-2 and 5-LOX, respectively, leading to highly predictive CoMFA models constructed on the basis of the binding conformations with q2 values of 0.782 and 0.634 for COX-2 and 5-LOX, respectively. In addition, CoMFA field distributions were found in good agreement with the structural characteristics of the corresponding binding sites. Both the docking simulations and QSAR analyses suggest that new potent dual inhibitors should share a structural feature with a moderately bulky group at R2 position and a rather negatively charged group around the position of the carbonyl group of DHDMBFs. Therefore, the final 3D-QSAR models and the information of the inhibitor-enzyme interaction should be useful in developing new NSAIDs as anti-inflammation drugs with favorable safety profile.     

Synthesis and biological evaluation of 1,3-diphenylprop-2-yn-1-ones as dual inhibitors of cyclooxygenases and lipoxygenases

A new class of 1,3-diphenylprop-2-yn-1-ones possessing a p-MeSO2 COX-2 phamacophore on the C-3 phenyl ring was designed for evaluation as dual inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX). Among the group of compounds evaluated, 1-(4-fluorophenyl)-3-(4-methanesulfonylphenyl)prop-2-yn-1-one (11j) exhibited excellent COX-2 inhibitory potency (COX-2 IC50 = 0.1 microM) and selectivity (SI = 300), whereas 1-(4-cyanophenyl)-3-(4-methanesulfonylphenyl)prop-2-yn-1-one (11d) exhibited an optimal combination of COX and LOX inhibition (COX-2 IC50 = 1.0 microM; COX-2 SI = 31.5; 5-LOX IC50 = 1.0 microM; 15-LOX IC50 = 3.2 microM).     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

COX-2/12-LOX dual pathway, a novel strategy for treatment of multiple myeloma?

Cyclooxygenase (COX) and lipoxygenase (LOX) metabolic enzymes are the two main pathways for arachidonic acid (AA) metabolism. Emerging reports now indicate alterations of arachidonic acid metabolism with carcinogenesis and many COX and LOX inhibitors are being investigated as potential anticancer drugs. COX-2 is frequently expressed in many tumors, such as multiple myeloma (MM), a disorder in which malignant plasma cells accumulate, generally derived from one clone in the bone marrow, and is an independent predictor of poor outcome. 12-LOX, an important member of LOX, is proved to be expressed in MM cells. We hypothesize that COX-2 and 12-LOX represent an integrated system, COX-2/12-LOX dual pathway, which much more efficiently enhances the intracellular levels of unesterified arachidonate and regulates cell proliferative, apoptosis and pro-angiogenic potential of MM. The COX-2/12-LOX dual pathway may act as a novel potential strategy for treatment of tumors co-expressing COX-2 and 12-LOX, and the agents that can simultaneously inhibit the two enzymes of COX-2 and 12-LOX may present a novel and promising therapeutic approach to these tumors.     

Synthesis and biological evaluation of a novel class of rofecoxib analogues as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs)

A group of 4-(4-methanesulfonylphenyl)-3-phenyl-2(5H)furanones possessing an acetyl, 3-oxobut-1-ynyl, [hydroxyl(or alkoxy)imino]alkyl, [hydroxyl(or alkoxy)imino]alkynyl, and N-alkoxy(or N-phenoxy)carbonyl-N-hydroxy-N-ethylamino substituents at the para-position of the C-3 phenyl ring of rofecoxib were synthesized. This group of compounds was designed for evaluation as dual inhibitors of cyclooxygenases (COXs) and lipoxygenases (LOXs) that exhibit in vivo anti-inflammatory and analgesic activities. In vitro COX-1/COX-2, and 5-LOX/15-LOX, isozyme inhibition structure-activity relationships identified 3-[4-(1-hydroxyimino)ethylphenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (17a) having an optimal combination of COX-2 (COX-2 IC50 = 1.4 microM; COX-2 SI > 71), and 5-LOX and 15 LOX (5-LOX IC50 = 0.28 microM; 15-LOX IC50 = 0.32 microM), inhibitory effects. It was also discovered that 3-[4-(3-hydroxyiminobut-1-ynyl)phenyl]-4-(4-methanesulfonylphenyl)-2(5H)furanone (18a) possesses dual COX-2 (IC50 = 2.7 microM) and 5-LOX (IC50 = 0.30 microM) inhibitor actions. Further in vivo studies employing a rat carrageenan-induced paw edema model showed that the oxime compounds (17a, 18a) were more potent anti-inflammatory agents than the 5-LOX inhibitor caffeic acid, and 15-LOX inhibitor nordihydroguaiaretic acid (NDGA), but less potent than the selective COX-2 inhibitor celecoxib. The results of this investigation showed that incorporation of a para-oxime moiety on the C-3 phenyl ring of rofecoxib provides a suitable template for the design of dual inhibitors of the COX and LOX enzymes.     

Computer-aided drug design guided by hydrogen/deuterium exchange mass spectrometry: A powerful combination for the development of potent and selective inhibitors of Group VIA calcium-independent phospholipase A2

Potent and selective inhibitors for phospholipases A₂ (PLA₂) are useful for studying their intracellular functions. PLA₂ enzymes liberate arachidonic acid from phospholipids activating eicosanoid pathways that involve cyclooxygenase (COX) and lipoxygenase (LOX) leading to inflammation. Anti-inflammatory drugs target COX and LOX; thus, PLA₂ can also be targeted to diminish inflammation at an earlier stage in the process. This paper describes the employment of enzymatic assays, hydrogen/deuterium exchange mass spectrometry (DXMS) and computational chemistry to develop PLA₂ inhibitors. Beta-thioether trifluoromethylketones (TFKs) were screened against human GVIA calcium-independent, GIVA cytosolic and GV secreted PLA₂s. These compounds exhibited inhibition toward Group VIA calcium-independent PLA₂ (GVIA iPLA₂), with the most potent and selective inhibitor 3 (OTFP) obtaining an X_I(50) of 0.0002 mole fraction (IC₅₀ of 110 nM). DXMS binding experiments in the presence of OTFP revealed the peptide regions of GVIA iPLA₂ that interact with the inhibitor. Molecular docking and dynamics simulations in the presence of a membrane were guided by the DXMS data in order to identify the binding mode of OTFP. Clustering analysis showed the binding mode of OTFP that occupied 70% of the binding modes occurring during the simulation. The resulted 3D complex was used for docking studies and a structure–activity relationship (SAR) was established. This paper describes a novel multidisciplinary approach in which a 3D complex of GVIA iPLA₂ with an inhibitor is reported and validated by experimental data. The SAR showed that the sulfur atom is vital for the potency of beta-thioether analogues, while the hydrophobic chain is important for selectivity. This work constitutes the foundation for further design, synthesis and inhibition studies in order to develop new beta-thioether analogues that are potent and selective for GVIA iPLA₂ exclusively.     

Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species

We show that rat neonatal cardiac myocytes exposed to 1 micromol/l of the calcium ionophore A23187 respond with an enhanced production of reactive oxygen species (ROS). This dose is not cytotoxic to the myocytes. A higher concentration (10 micromol/l) evokes less ROS production and is significantly cytotoxic 24 h after exposure, but not immediately after removal of the A23187, when ROS are measured. Both cell death and the decrease in mitochondrial potential are only partially sensitive to MPT inhibitor cyclosporin A. Experiments performed to elucidate the sources of ROS included use of the nitric oxide synthase (NOS) inhibitor L-NAME; NOS involvement was excluded. Experiments with the oxidative phosphorylation uncoupler CCCP revealed that mitochondria are at least partially responsible for the observed effect. Further studies with cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors (indomethacin and MK886, respectively) showed that these enzymes could also be sources of ROS when the calcium level is elevated. Their effect appeared to be independent of phospholipase A(2) inhibition, suggesting that COX and LOX stimulation is not due to elevated substrate (arachidonic acid) concentration but rather to a direct effect of calcium.     

Synthesis, biological evaluation and molecular modeling study of pyrazole and pyrazoline derivatives as selective COX-2 inhibitors and anti-inflammatory agents. Part 2

New pyrazole and pyrazoline derivatives have been synthesized and their ability to inhibit ovine COX-1/COX-2 isozymes was evaluated using in vitro cyclooxygenase (COX) inhibition assay. Among the tested compounds, N-((5-(4-chlorophenyl)-1-phenyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)methylene)-3,5-bis(trifluoromethyl)aniline 8d exhibit optimal COX-2 inhibitory potency (IC(50)=0.26 lM) and selectivity (SI)=>192.3] comparable with reference drug celecoxib (IC(50) value of 0.28 lM and selectivity index of 178.57). Moreover, the anti-inflammatory activity of selected compounds, which are the most selective COX-2 inhibitors in the COX inhibition assay, was investigated in vivo using carrageenan-induced rat paw edema model. Molecular modeling was conducted to study the ability of the active compounds to bind into the active site of COX-2 which revealed a similar binding mode to SC-558, a selective COX-2 inhibitor.     

Effects of cyclooxygenase and lipoxygenase inhibition on basal- and serotonin-induced ion transport in rat colon

The purpose of this study was to determine the effect of a selective cyclooxygenase (COX)-2 inhibitor as compared to non-selective COX and lipoxygenase (LOX) inhibitors in rat colon. Basal- and serotonin (5-hydroxytryptamine, 5-HT)-induced electrogenic ion transport (short circuit current, SCC), prostaglandin E2 (PGE2) release and histological characteristics were measured. Muscle-stripped mucosal sheets of the proximal and distal segment of rat colon were investigated by employing the Ussing chamber technique, radioimmunoassays for PGE2 and light microscopy examinations for control of tissue integrity. 5-HT and PGE2 both induced a concentration-dependent increase in SCC by activation of multiple receptors. The response to 5-HT was bumetanide-sensitive. Neither the non-selective COX inhibitor piroxicam, nor the selective COX-2 inhibitor SC-'236, altered basal- SCC or 5-HT-induced SCC. Indomethacin reduced both basal- and 5-HT-induced SCC in both segments. Nordihydroguaiaretic acid reduced the 5-HT-induced increase in SCC, but did not change basal SCC. 5-HT-induced a concentration-dependent release of PGE2. Only high concentrations of piroxicam and indomethacin reduced basal PGE2 release and 5-HT-induced PGE2 release. Histological examination of the specimens demonstrated only minor changes following mounting in chambers. There were no apparent differences in the morphology following treatment with COX or LOX inhibitors. These results suggest that in rat colon only the COX-1 enzyme is expressed under basal conditions. Furthermore, data suggest neither the COX-1 nor the COX-2 enzyme to be of major importance for 5-HT-induced ion transport in rat colon in vitro. In conclusion, this study supports 5-HT as a mediator of chloride secretion by activating several receptor subtypes and the LOX enzyme, releasing mediators such as leucotrienes.     

Design,synthesis and structure–activity relationship studies of morpholino-1H-phenalene derivatives that antagonize Mcl-1/Bcl-2

We report herein characteristic studies of Mcl-1 and Bcl-2 dual inhibitors. It was found that a protruding carbonyl group forming hydrogen bond with R263 plays a predominant role compared with the hydrophobic group that occupies the p2 pocket. A series of dual inhibitors representing different parts of the morpholino-1H-phenalene were designed, synthesized and evaluated.     

Purification, product characterization and kinetic properties of lipoxygenase from olive fruit (Olea europaea L.).

Lipoxygenase from olive fruit was purified to homogeneity for the first time after differential centrifugations and by hydrophobic chromatography. The enzyme had a molecular mass of 98 kDa and exhibited a maximal activity at pH 6. Lipoxygenase had a better affinity for linoleic acid (Km=82.44 microM) than for linolenic acid (Km = 306.26 microM). It is inhibited by linoleate:oxygen oxidoreductase (LOX) inhibitors like nordihydroguaiaretic acid (NDGA) or propyl gallate. The reaction product was 13-hydroperoxy octadecadienoic acid when linoleic acid was used as substrate.     

Binding of alkylurea inhibitors to epoxide hydrolase implicates active site tyrosines in substrate activation

The structures of two alkylurea inhibitors complexed with murine soluble epoxide hydrolase have been determined by x-ray crystallographic methods. The alkyl substituents of each inhibitor make extensive hydrophobic contacts in the soluble epoxide hydrolase active site, and each urea carbonyl oxygen accepts hydrogen bonds from the phenolic hydroxyl groups of Tyr(381) and Tyr(465). These hydrogen bond interactions suggest that Tyr(381) and/or Tyr(465) are general acid catalysts that facilitate epoxide ring opening in the first step of the hydrolysis reaction; Tyr(465) is highly conserved among all epoxide hydrolases, and Tyr(381) is conserved among the soluble epoxide hydrolases. In one enzyme-inhibitor complex, the urea carbonyl oxygen additionally interacts with Gln(382). If a comparable interaction occurs in catalysis, then Gln(382) may provide electrostatic stabilization of partial negative charge on the epoxide oxygen. The carboxylate side chain of Asp(333) accepts a hydrogen bond from one of the urea NH groups in each enzyme-inhibitor complex. Because Asp(333) is the catalytic nucleophile, its interaction with the partial positive charge on the urea NH group mimics its approach toward the partial positive charge on the electrophilic carbon of an epoxide substrate. Accordingly, alkylurea inhibitors mimic features encountered in the reaction coordinate of epoxide ring opening, and a structure-based mechanism is proposed for leukotoxin epoxide hydrolysis.     

Synthesis and biological evaluation of 2,5-disubstituted 1,3,4-oxadiazole derivatives with both COX and LOX inhibitory activity

Dual cyclooxygenase/lipoxygenase (COX/LOX) inhibitors constitute a valuable alternative to classical nonsteroidal anti-inflammatory drugs (NSAIDs) and selective COX-2 inhibitors for the treatment of inflammatory diseases. A series of 3-(5-phenyl/phenylamino-[1,3,4]oxadiazol-2-yl)-chromen-2-one and N-[5-(2-oxo-2H-chromen-3-yl)-[1,3,4]oxadiazol-2-yl]-benzamide derivatives were synthesized and screened for anti-inflammatory, analgesic activity. All the derivatives prepared are active in inhibiting oedema induced by carrageenan. Compound 4e was found more potent with 89% of inhibition followed by compound 4b (86%). Compounds with >70% of anti-inflammatory activity were tested for analgesic, ulcerogenic, and lipid peroxidation profile. Selected compounds were also evaluated for inhibition of COXs (COX-1 and COX-2) and LOXs (LOX-5, LOX-12, and LOX-15). Compound 4e was comparatively selective for COX-2, LOX-5, and LOX-15. Study revealed that these derivatives were more effective than ibuprofen with reduced side effects. It can be suggested that these derivatives could be used to develop more potent and safer NSAIDs.