Hydroperoxide bicyclase CYP50918A1 of Plasmodiophora brassicae (Rhizaria, SAR): Detection of novel enzyme of oxylipin biosynthesis.

The genome of the cabbage clubroot pathogen Plasmodiophora brassicae Woronin 1877 (Cercozoa, Rhizaria, SAR), possesses two expressed genes encoding the P450s that are phylogenetically related to the enzymes of oxylipin biosynthesis of the CYP74 clan. The cDNA of one of these genes (CYP50918A1) has been expressed in E. coli. The preferred substrate for the recombinant protein, the 13-hydroperoxide of α-linolenic acid (13-HPOT), was converted to the novel heterobicyclic oxylipins, plasmodiophorols A and B (1 and 2) at the ratio ca. 12:1. Compounds 1 and 2 were identified as the substituted 6-oxabicyclo[3.1.0]hexane and 2-oxabicyclo[2.2.1]heptane (respectively) using the MS and NMR spectroscopy, as well as the chemical treatments. The 18O labelling experiments revealed the incorporation of a single 18O atom from [18O2]13-HPOT into the epoxide and ether functions of products 1 and 2 (respectively), but not into their OH groups. In contrast, the 18O from [18O2]water was incorporated only into the hydroxyl functions. One more minor polar product, plasmodiophorol C (3), identified as the cyclopentanediol, was formed through the hydrolysis of compounds 1 and 2. Plasmodiophorols A-C are the congeners of egregiachlorides, hybridalactone, ecklonialactones and related bicyclic oxylipins detected before in some brown and red algae. The mechanism of 13-HPOT conversions to plasmodiophorols A and B involving the epoxyallylic cation intermediate is proposed. The hydroperoxide bicyclase CYP50918A1 is the first enzyme controlling this kind of fatty acid hydroperoxide conversion.

Synthesis of New Tricyclic 1,2-Thiazine Derivatives with Anti-Inflammatory Activity.

New, tricyclic compounds containing a sulfonyl moiety in their structure, as potential safer COX inhibitors, were designed and synthesized. New derivatives have three conjugated rings and a sulfonyl group. A third ring, i.e., an oxazine, oxazepine or oxazocin, has been added to the 1,2-benzothiazine skeleton. Their anti-COX-1/COX-2 and cytotoxic effects in vitro on NHDF cells, together with the ability to interact with model membranes and the influence on reactive oxygen species and nitric oxide, were studied. Additionally, a molecular docking study was performed to understand the binding interaction of the compounds with the active site of cyclooxygenases. For the abovementioned biological evaluation of new tricyclic 1,2-benzothiazine derivatives, the following techniques and procedures were employed: the differential scanning calorimetry, the COX colorimetric inhibitor screening assay, the MTT, DCF-DA and Griess assays. All of the compounds studied demonstrated preferential inhibition of COX-2 compared to COX-1. Moreover, all the examined tricyclic 1,2-thiazine derivatives interacted with the phospholipid model membranes. Finally, they neither have cytotoxic potency, nor demonstrate significant influence on the level of reactive oxygen species or nitric oxide. Overall, the tricyclic 1,2-thiazine derivatives are good starting points for future pharmacological tests as a group of new anti-inflammatory agents.

A New N-Substituted 1H-Isoindole-1,3(2H)-Dione Derivative-Synthesis, Structure and Affinity for Cyclooxygenase Based on In Vitro Studies and Molecular Docking.

Isoindoline-1,3-dione derivatives constitute an important group of medicinal substances. In this study, nine new 1H-isoindole-1,3(2H)-dione derivatives and five potential pharmacophores were obtained in good yield (47.24-92.91%). The structure of the new imides was confirmed by the methods of elemental and spectral analysis: FT-IR, H NMR, and MS. Based on the obtained results of ESI-MS the probable path of the molecules decay and the hypothetical structure of the resulting pseudo-molecular ions have been proposed. The physicochemical properties of the new phthalimides were determined on the basis of Lipinski's rule. The biological properties were determined in terms of their cyclooxygenase (COX) inhibitory activity. Three compounds showed greater inhibition of COX-2, three compounds inhibited COX-1 more strongly than the reference compound meloxicam. From the obtained results, the affinity ratio COX-2/COX-1 was calculated. Two compounds had a value greater than that of meloxicam. All tested compounds showed oxidative or nitrosan stress (ROS and RNS) scavenging activity. The degree of chromatin relaxation outside the cell nucleus was lower than the control after incubation with all test compounds. The newly synthesized phthalimide derivatives showed no cytotoxic activity in the concentration range studied (10-90 µM). A molecular docking study was used to determined interactions inside the active site of cyclooxygenases.

Bioactive stigmastadienone from Isodon rugosus as potential anticholinesterase, α-glucosidase and COX/LOX inhibitor: In-vitro and molecular docking studies.

Natural product is a well-known source of bioactive compounds. Herein, a steroidal compound stigmasta-7,22-diene-3-one (stigmastadienone) has been isolated from Isodon rugosus. The potency of isolated compound has been tested for several in-vitro targets. The acetyl and butyrylcholinesterase assays were performed using Ellman's procedure. For the in-vitro antidiabetic potential, α-glucosidase inhibitory assay was performed. Similarly, the cyclo and lipoxygenase pathways were studied to find its potential role in the management of inflammation and analgesia. The 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and hydrogen peroxide (H2O2) assays were performed for the antioxidant potentials. Docking studies were performed against acetylcholinesterase, cyclooxygenase and lipoxygenase targets. In anticholinesterase assays, stigmastadienone exhibited half-maximal inhibitory concentration (IC50) values of 13.52 and 11.53 µg/ml for acetyl and butyrylcholinesterase respectively. The observed IC50 values for that of galantamine were 6.07 and 4.42 µg/ml for acety and butyrylcholinesterase respectively. In inhibiting α-glucosidase enzyme, the compound showed mediocre IC50 of 109.40 µg/ml compared to the standard acarbose (7.60 µg/ml). The stigmastadienone proved to be an excellent inhibitor of cyclooxygenase 2 (COX-2) and 5-lipoxygenase (5-LOX) attaining IC50 values of 4.72 and 3.36 µg/ml respectively. The standard drugs IC50 values for COX-2 (celecoxib) and 5-LOX (montelukast) were 3.81 and 2.74 µg/ml respectively. The enzymatic activities of stigmastadienone were also supplemented with antioxidant results, specifically it was more dominant against DPPH and ABTS free radicals. Docking studies showed that only the carbonyl oxygen is able to form hydrogen bond interaction with the residues. In conclusions, the stigmastadienone has been isolated from Isodon rugosus for the first time. Moreover, the compound has been evaluated for several biochemical pathways which suggest its pharmacological role on the explored targets.

Biochemical characterization of the cyclooxygenase enzyme in penaeid shrimp.

Cyclooxygenase (COX) is a two-step enzyme that converts arachidonic acid into prostaglandin H2, a labile intermediate used in the production of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α). In vertebrates and corals, COX must be N-glycosylated on at least two asparagine residues in the N-(X)-S/T motif to be catalytically active. Although COX glycosylation requirement is well-characterized in many species, whether crustacean COXs require N-glycosylation for their enzymatic function have not been investigated. In this study, a 1,842-base pair cox gene was obtained from ovarian cDNA of the black tiger shrimp Penaeus monodon. Sequence analysis revealed that essential catalytic residues and putative catalytic domains of P. monodon COX (PmCOX) were well-conserved in relation to other vertebrate and crustacean COXs. Expression of PmCOX in 293T cells increased levels of secreted PGE2 and PGF2α up to 60- and 77-fold, respectively, compared to control cells. Incubation of purified PmCOX with endoglycosidase H, which cleaves oligosaccharides from N-linked glycoproteins, reduced the molecular mass of PmCOX. Similarly, addition of tunicamycin, which inhibits N-linked glycosylation, in PmCOX-expressing cells resulted in PmCOX protein with lower molecular mass than those obtained from untreated cells, suggesting that PmCOX was N-glycosylated. Three potential glycosylation sites of PmCOX were identified at N79, N170 and N424. Mutational analysis revealed that although all three residues were glycosylated, only mutations at N170 and N424 completely abolished catalytic function. Inhibition of COX activity by ibuprofen treatment also decreased the levels of PGE2 in shrimp haemolymph. This study not only establishes the presence of the COX enzyme in penaeid shrimp, but also reveals that N-glycosylation sites are highly conserved and required for COX function in crustaceans.

Nanocluster-assisted protein-film voltammetry for direct electrochemical signal acquisition.

Acquisition of the direct electrochemical response of protein is the cornerstone for the development of the third generation of electrochemical biosensors. In this work, we developed a nanocluster-assisted protein-film voltammetry technique (NCA-PFV) which can achieve the acquisition of the electrochemical signal and maintain the activity without affecting of the protein's structure. With this strategy, a lipid bilayer membrane is used to immobilize the membrane protein so as to maintain its natural state. Copper nanoclusters with a size smaller than most proteins are then used to function at sub-protein scale and to mediate the electron hopping from the electroactive center of the electrode. As a model, the direct electrochemical signal of cyclooxygenase (COX) is successfully obtained, with a pair of well-defined redox peaks located at -0.39 mV and -0.31 mV, which characterize the heme center of the enzyme. Its catalytic activity towards the substrate arachidonic acid (AA) is also retained. The detection range for AA is 10-1000 µM and the detection limit is 2.4 µM. Electrochemical monitoring of the regulation of the catalytic activity by an inhibitor DuP-697 is also achieved. This work provides a powerful tool for the fabrication of enzyme-based electrochemical biosensors, and is also of great significance for promoting the development and application of next-generation electrochemical biosensors.

Modulation of Enzyme-Catalyzed Synthesis of Prostaglandins by Components Contained in Kidney Microsomal Preparations.

In the kidney, prostaglandins formed by cyclooxygenase 1 and 2 (COX-1 and COX-2) play an important role in regulating renal blood flow. In the present study, we report our observations regarding a unique modulatory effect of renal microsomal preparation on COX-1/2-mediated formation of major prostaglandin (PG) products in vitro. We found that microsomes prepared from pig and rat kidneys had a dual stimulatory-inhibitory effect on the formation of certain PG products catalyzed by COX-1 and COX-2. At lower concentrations, kidney microsomes stimulated the formation of certain PG products, whereas at higher concentrations, their presence inhibited the formation. Presence of kidney microsomes consistently increased the Km values of the COX-1/2-mediated reactions, while the Vmax might be increased or decreased depending on stimulation or inhibition observed. Experimental evidence was presented to show that a protein component present in the pig kidney microsomes was primarily responsible for the activation of the enzyme-catalyzed arachidonic acid metabolism leading to the formation of certain PG products.

Mechanism for the reactivation of the peroxidase activity of human cyclooxygenases: investigation using phenol as a reducing cosubstrate.

It has been known for many years that the peroxidase activity of cyclooxygenase 1 and 2 (COX-1 and COX-2) can be reactivated in vitro by the presence of phenol, which serves as a reducing compound, but the underlying mechanism is still poorly understood. In the present study, we use phenol as a model compound to investigate the mechanism by which the peroxidase activity of human COXs is reactivated after each catalytic cycle. Molecular docking and quantum mechanics calculations are carried out to probe the interaction of phenol with the peroxidase site of COXs and the reactivation mechanism. It is found that the oxygen atom associated with the Fe ion in the heme group (i.e., the complex of Fe ion and porphyrin) of COXs can be removed by addition of two protons. Following its removal, phenol can readily bind inside the peroxidase active sites of the COX enzymes, and directly interact with Fe in heme to facilitate electron transfer from phenol to heme. This investigation provides theoretical evidence for several intermediates formed in the COX peroxidase reactivation cycle, thereby unveiling mechanistic details that would aid in future rational design of drugs that target the peroxidase site.

Structural and Chemical Biology of the Interaction of Cyclooxygenase with Substrates and Non-Steroidal Anti-Inflammatory Drugs.

Cyclooxgenases are key enzymes of lipid signaling. They carry out the first step in the production of prostaglandins, important mediators of inflammation, pain, cardiovascular disease, and cancer, and they are the molecular targets for nonsteroidal anti-inflammatory drugs, which are among the oldest and most chemically diverse set of drugs known. Homodimeric proteins that behave as allosterically modulated, functional heterodimers, the cyclooxygenases exhibit complex kinetic behavior, requiring peroxide-dependent activation and undergoing suicide inactivation. Due to their important physiological and pathophysiological roles and keen interest on the part of the pharmaceutical industry, the cyclooxygenases have been the focus of a vast array of structural studies, leading to the publication of over 80 crystal structures of the enzymes in complex with substrates or inhibitors supported by a wealth of functional data generated by site-directed mutation experiments. In this review, we explore the chemical biology of the cyclooxygenases through the lens of this wealth of structural and functional information. We identify key structural features of the cyclooxygenases, break down their active site into regional binding pockets to facilitate comparisons between structures, and explore similarities and differences in the binding modes of the wide variety of ligands (both substrates and inhibitors) that have been characterized in complex with the enzymes. Throughout, we correlate structure with function whenever possible. Finally, we summarize what can and cannot be learned from the currently available structural data and discuss the critical intriguing questions that remain despite the wealth of information that has been amassed in this field.

Oral Intake of EPA:DHA 6:1 by Middle-Aged Rats for One Week Improves Age-Related Endothelial Dysfunction in Both the Femoral Artery and Vein: Role of Cyclooxygenases.

In humans, aging is associated with endothelial dysfunction and an increased risk of venous thromboembolism. Although intake of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) at a ratio of 6:1 by old rats improved the endothelial dysfunction in arteries, the impact on veins remains unclear. Eight-month-old male Wistar rats were either untreated or orally administered corn oil, EPA:DHA 1:1, or EPA:DHA 6:1 (500 mg/kg/d) for seven days. Vascular reactivity was studied by myography. In middle-aged femoral artery rings, acetylcholine caused a partial relaxation at low concentrations and a contractile response at high concentrations, whereas in the old femoral vein only a partial relaxation was observed. The EPA:DHA 6:1 treatment blunted the contractile response to acetylcholine in the middle-aged femoral artery and both EPA:DHA 6:1 and 1:1 increased the relaxation to acetylcholine in the old femoral vein. No such effects were observed with corn oil. Both the non-selective cyclooxygenase inhibitor indomethacin and the COX-1 inhibitor SC-560 increased the relaxation to acetylcholine in the middle-aged femoral artery whereas the COX-2 inhibitor NS-398 increased that in the middle-aged femoral vein. In conclusion, our results indicate that aging is associated with an endothelial dysfunction in the femoral artery and vein, which can be improved by EPA:DHA 6:1 treatment-most likely via a cyclooxygenase-dependent mechanism.

Pharmaco-Genetic Screen To Uncover Actin Regulators Targeted by Prostaglandins During Drosophila Oogenesis.

Prostaglandins (PGs) are lipid signaling molecules with numerous physiologic functions, including pain/inflammation, fertility, and cancer. PGs are produced downstream of cyclooxygenase (COX) enzymes, the targets of non-steroidal anti-inflammatory drugs (NSAIDs). In numerous systems, PGs regulate actin cytoskeletal remodeling, however, their mechanisms of action remain largely unknown. To address this deficiency, we undertook a pharmaco-genetic interaction screen during late-stage Drosophila oogenesis. Drosophila oogenesis is as an established model for studying both actin dynamics and PGs. Indeed, during Stage 10B, cage-like arrays of actin bundles surround each nurse cell nucleus, and during Stage 11, the cortical actin contracts, squeezing the cytoplasmic contents into the oocyte. Both of these cytoskeletal properties are required for follicle development and fertility, and are regulated by PGs. Here we describe a pharmaco-genetic interaction screen that takes advantage of the fact that Stage 10B follicles will mature in culture and COX inhibitors, such as aspirin, block this in vitro follicle maturation. In the screen, aspirin was used at a concentration that blocks 50% of the wild-type follicles from maturing in culture. By combining this aspirin treatment with heterozygosity for mutations in actin regulators, we quantitatively identified enhancers and suppressors of COX inhibition. Here we present the screen results and initial follow-up studies on three strong enhancers - Enabled, Capping protein, and non-muscle Myosin II Regulatory Light Chain. Overall, these studies provide new insight into how PGs regulate both actin bundle formation and cellular contraction, properties that are not only essential for development, but are misregulated in disease.

Structure and Dynamics of the Acyl Chains in the Membrane Trafficking and Enzymatic Processing of Lipids.

The regulatory chemical mechanisms of lipid trafficking and degradation are involved in many pathophysiological processes, being implicated in severe pain, inflammation, and cancer. In addition, the processing of lipids is also relevant for industrial and environmental applications. However, there is poor understanding of the chemical features that control lipid membrane trafficking and allow lipid-degrading enzymes to efficiently select and hydrolyze specific fatty acids from a complex cellular milieu of bioactive lipids. This is particularly true for lipid acyl chains, which have diverse structures that can critically affect the many complex reactions needed to elongate, desaturate, or transport fatty acids. Building upon our own contributions in this field, we will discuss how molecular simulations, integrated with experimental evidence, have revealed that the structure and dynamics of the lipid tail are actively involved in modulating membrane trafficking at cellular organelles, and enzymatic reactions at cell membranes. Further evidence comes from recent crystal structures of lipid receptors and remodeling enzymes. Taken together, these recent works have identified those structural features of the lipid acyl chain that are crucial for the regioselectivity and stereospecificity of essential desaturation reactions. In this context, we will first illustrate how atomistic and coarse-grained simulations have elucidated the structure-function relationships between the chemical composition of the lipid's acyl chains and the molecular properties of lipid bilayers. Particular emphasis will be given to the prominent chemical role of the number of double carbon-carbon bonds along the lipid acyl chain, that is, discriminating between saturated, monounsaturated, and polyunsaturated lipids. Different levels of saturation in fatty acid molecules dramatically influence the biophysical properties of lipid assemblies and their interaction with proteins. We will then discuss the processing of lipids by membrane-bound enzymes. Our focus will be on lipids such as anandamide and 2-arachidonoylglycerol. These are the main molecules that act as neurotransmitters in the endocannabinoid system. Specifically, recent findings indicate a crucial interplay between the level of saturation of the lipid tail, its energetically and sterically favored conformations, and the hydrophobic accessory cavities in lipid-degrading enzymes, which help form catalytically active conformations of the selected substrate. This Account will emphasize how the specific chemical structure of acyl chains affects the molecular mechanisms for modulating membrane trafficking and selective hydrolysis. The results examined here show that, by using molecular simulations to investigate lipid plasticity and substrate flexibility, researchers can enrich their interpretation of experimental results about the structure-function relationships of lipids. This could positively impact chemical and biological studies in the field and ultimately support protein engineering studies and structure-based drug discovery to target lipid-processing enzymes.

Profiling the interaction of 1-phenylbenzimidazoles to cyclooxygenases.

In the design of 1-phenylbenzimidazoles as model cyclooxygenase (COX) inhibitors, docking to a series of crystallographic COX structures was performed to evaluate their potential for high-affinity binding and to reproduce the interaction profile of well-known COX inhibitors. The effect of ligand-specific induced fit on the calculations was also studied. To quantitatively compare the pattern of interactions of model compounds to the profile of several cocrystallized COX inhibitors, a geometric parameter, denominated ligand-receptor contact distance (LRCD), was developed. The interaction profile of several model complexes showed similarity to the profile of COX complexes with inhibitors such as iodosuprofen, iodoindomethacin, diclofenac, and flurbiprofen. Shaping of high-affinity binding sites upon ligand-specific induced fit mostly determined both the affinity and the binding mode of the ligands in the docking calculations. The results suggest potential of 1-phenylbenzimidazole derivatives as COX inhibitors on the basis of their predicted affinity and interaction profile to COX enzymes. The analyses also provided insights into the role of induced fit in COX enzymes. While inhibitors produce different local structural changes at the COX ligand binding site, induced fit allows inhibitors in diverse chemical classes to share characteristic interaction patterns that ensure key contacts to be achieved. Different interaction patterns may also be associated with different inhibitory mechanisms.

Celecoxib for the treatment of musculoskeletal arthritis.

Introduction: The cyclooxygenase (COX)-2 inhibitor celecoxib is an approved compound for rheumatoid (RA) and osteoarthritis (OA), combining both anti-inflammatory and analgesic properties with a good gastrointestinal tolerability. Areas covered: This article covers the pharmacological properties and clinical efficacy as well as the latest safety data available for celecoxib with emphasis on the treatment of RA and OA. It is based primarily on a current literature search on PubMed and Web of Science, but also on the professional rheumatological expertise of the authors. Expert opinion: Celecoxib has been shown to be superior to placebo and equivalent to traditional non-steroidal anti-inflammatory drugs (tNSAIDs). Many studies have been published making celecoxib a good and safe treatment option in particular in moderate arthritis and patients without established cardiovascular (CV) disease. Moreover, older patients might gain significant benefits compared to tNSAIDs due to reduced gastrointestinal events even when having a history of ulcer bleedings. Nonetheless, there is still much to learn, especially regarding the prescription of celecoxib in patients with cardiovascular co-morbidities. While low doses seem to be safe according to present data, the knowledge on the more effective, higher doses >400 mg/day is still limited.

Albizia anthelmintica: HPLC-MS/MS profiling and in vivo anti-inflammatory, pain killing and antipyretic activities of its leaf extract.

In the current work, the phytochemical composition of a leaf methanol extract from Albizia anthelmintica was thoroughly investigated. The antioxidant, anti-inflammatory, analgesic, and antipyretic activities of the extract were investigated. In the carrageenan induced hind paw edema bioassay; the extract significantly reduced the edema thickness in rats and diminished the leukocyte migration to the peritoneal cavity in mice. The extract exhibited central and peripheral anti-nociceptive effects; it significantly decreased the number of acetic acid induced writhes and prolonged the latency time in the hot plate test. The extract showed a substantial antipyretic activity as it decreased significantly the elevated rectal temperature in mice after intraperitoneal injection of Brewer's yeast. Molecular docking of some major compounds in the extract to COX-1, COX-2 and 5-LOX, enzymes involved in the inflammation cascade, revealed appreciable interactions with the conserved amino acid residues in these target proteins. These findings were confirmed with in vitro enzyme inhibitory assays in which the extract showed IC50 values of 4.11, 0.054, and 1.74 µg/mL towards COX-1, COX-2 and 5-LOX, respectively. The extract displayed solid antioxidant properties as well with a TAC value of 35.13 U/L and EC50of 5.36 µg/mL in DPPH assay. These findings suggested that Albizia anthelmintica is a good antioxidant with potential therapeutic efficacy for treating inflammation, pain and related oxidative stress disorders.

Synergistically dual-functional nano eye-drops for simultaneous anti-inflammatory and anti-oxidative treatment of dry eye disease.

We have developed a rapid and straightforward topical treatment method for dry eye disease (DED) using poly(catechin) capped-gold nanoparticles (Au@Poly-CH NPs) carrying amfenac [AF; a nonsteroidal anti-inflammatory drug (NSAID)] through effective attenuation of ocular surface tissue damage in dry eyes. A dual-targeted strategy based on ocular therapeutics was adopted to simultaneously block the cyclooxygenase enzymes-induced inflammation and reactive oxygen species (ROS)-induced oxidative stress, the primary two causes of DED. The self-assembled core-shell Au@Poly-CH NPs synthesized via a simple reaction between tetrachloroaurate(iii) and catechin possess a poly(catechin) shell (∼20 nm) on the surface of each Au NP (∼60 nm). The anti-oxidant and anti-inflammatory properties of AF/Au@Poly-CH NPs were evaluated by DCFH-DA and prostaglandin E2/VEGF assays, respectively. Our results demonstrate that Au@Poly-CH NPs not only act as an anti-oxidant to suppress ROS-mediated processes, but also serve as a drug carrier of AF for a synergistic effect on anti-inflammation. In vivo biocompatibility studies show good tolerability of AF/Au@Poly-CH NPs for potential use in the treatment of ocular surface pathologies. The dual-targeted therapeutic effects of AF/Au@Poly-CH NPs lead to rapid recovery from DED in a rabbit model. Au@Poly-CH NPs loaded with NSAIDs is a promising multifunctional nanocomposite for treating various inflammation- and oxidative stress-related diseases.

Optimization of structures, biochemical properties of ketorolac and its degradation products based on computational studies.

BACKGROUND: Ketorolac (KTR) is used as an analgesic drug with an efficacy close to that of the opioid family. It is mainly used for the short term treatment of post-operative pain. It can inhibit the prostaglandin synthesis by blocking cyclooxygenase (COX). METHODS: In this investigation, the inherent stability and biochemical interaction of Ketorolac (KTR) and its degradation products have been studiedon the basis of quantum mechanical approaches. Density functional theory (DFT) with B3LYP/ 6-31G (d) has been employed to optimize the structures. Thermodynamic properties, frontier molecular orbital features, dipole moment, electrostatic potential, equilibrium geometry, vibrational frequencies and atomic partial charges of these optimized structureswere investigated. Molecular docking has been performed against prostaglandin H2 (PGH2) synthase protein 5F19 to search the binding affinity and mode(s). ADMET prediction has performed to evaluate the absorption, metabolism and carcinogenic properties. RESULTS: The equilibrium geometry calculations support the optimized structures. Thermodynamic results disclosed the thermal stability of all structures. From molecular orbital data, all the degradents are chemically more reactive than parent drug (except K3). However, the substitution of carboxymethyl radicalin K4 improved the physicochemical properties and binding affinity. ADMET calculations predict the improved pharmacokinetic and non-carcinogenic properties of all degradents. CONCLUSION: Based on physicochemical, molecular docking, and ADMET calculation, this study can be helpful to understand the biochemical activities of Ketorolac and its degradents and to design a potent analgesic drug.

A crystallographic and theoretical study of an (E)-2-Hydroxyiminoethanone derivative: prediction of cyclooxygenase inhibition selectivity of stilbenoids by MM-PBSA and the role of atomic charge.

We recently reported that the hydroxyiminoethanone derivative, (E)-OXM, behaves as a highly selective COX-1 inhibitor (COX-1 SI = 833), and also an interesting scaffold with unique characteristics. In the current study, a comprehensive crystallographic and computational study was performed to elucidate its conformational stability and pharmacological activity. Its conformational energy was studied at the B3LYP/6-311G** level of theory and compared to the single-crystal X-ray diffraction data. In addition, computational studies of three structurally different stilbenoid derivatives used as selective COX-1 or COX-2 inhibitors were undertaken to predict their COX selectivity potentials. Flexible docking was performed for all compounds at the active site of both COX-1 and COX-2 enzymes by considering some of the key residues as flexible during the docking operation. In the next step, molecular dynamic simulation and binding free energy calculations were performed by MM-PBSA. Final results were found to be highly dependent on the atomic charges of the inhibitors and the choice of force field used to calculate the atomic charges. The binding conformation of the hydroxyiminoethanone derivative is highly correlated with the type of COX isoform inhibited. Our predictive approach can truly predict the cyclooxygenase inhibition selectivity of stilbenoid inhibitors.

Report- Role of prostaglandin H synthase in activation of acrylonitrile to cyanide.

The present study aimed at investigating the in-vitro oxidation of acrylonitrile (ACN) to cyanide (CN-) by prostaglandin H synthase (PHS). Detection of CN- is considered a marker for free radical intermediates involved in ACN-induced toxicity. First, most favorable circumstances for ACN oxidation were characterized: pH (4.5), temperature (37ºC) and time of incubation (60 min.). In addition, the concentrations of ACN, PHS and H2O2 in incubation mixtures were assessed for further reaction characterization. The reaction maximum velocity (Vmax) was calculated to be 582.75 pmol CN-/mL/min and the Michaelis-Menten constant (Km) was 149.25 µmol ACN. Adding PHS inhibitors; resveratrol, quercetin, indomethacin or troloc-C to the reaction mixtures significantly reduced the rate of ACN oxidation. In conclusion, the present study demonstrates the ability of PHS to oxidize ACN to CN- and provides a clue for the explanation of ACN target toxicity.