Molecular dynamics simulations of arachidonic acid complexes with COX-1 and COX-2: insights into equilibrium behavior

The cyclooxygenase (COX) enzymes are responsible for the committed step in prostaglandin biosynthesis, the generation of prostaglandin H(2). As a result, these enzymes are pharmacologically important targets for nonsteroidal antiinflammatory drugs, such as aspirin and newer COX-2 selective inhibitors. The cyclooxygenases are functional homodimers, and each subunit contains both a cyclooxygenase and a peroxidase active site. These enzymes are quite interesting mechanistically, as the conversion of arachidonic acid to prostaglandin H(2) requires two oxygenation and two cyclization reactions, resulting in the formation of five new chiral centers with nearly absolute regio- and stereochemical fidelity. We have used molecular dynamics (MD) simulations to investigate the equilibrium behavior of both COX-1 and COX-2 enzyme isoforms with bound arachidonate. These simulations were compared with reference simulations of arachidonate in solution to explore the effect of enzyme on substrate conformation and positioning in the active site. The simulations suggest that the substrate has greater conformational freedom in the COX-2 active site, consistent with the larger COX-2 active site volume observed in X-ray crystal structures. The simulations reveal different conformational behavior for arachidonate in each subunit over the course of extended equilibrium MD simulations. The simulations also provide detailed information for several protein channels that might be important for oxygen and water transport to or from active sites or for intermediate trafficking between the cyclooxygenase and peroxidase active sites. The detailed comparisons for COX-1 versus COX-2 active site structural fluctuations may also provide useful information for design of new isozyme-selective inhibitors.     

Evidence for a free radical mechanism of styrene-glutathione conjugate formation catalyzed by prostaglandin H synthase and horseradish peroxidase

We have proposed, using styrene as a model, a new mechanism for the formation of glutathione conjugates that is independent of epoxide formation but dependent on the oxidation of glutathione to a thiyl radical by peroxidases such as prostaglandin H synthase or horseradish peroxidase. The thiyl radical reacts with styrene to yield a carbon-centered radical which subsequently reacts with molecular oxygen to give the styrene-glutathione conjugate. We have used electron spin resonance spin trapping techniques to detect the proposed free radical intermediates. A styrene carbon-centered radical was trapped using the spin traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and t-nitrosobutane. The position of the carbon-centered radical was confirmed to be at carbon 7 by the use of specific 2H-labeled styrenes. The addition of the spin trap DMPO inhibited both the utilization of molecular oxygen and the formation of styrene-glutathione conjugates. Under anaerobic conditions additional styrene-glutathione conjugates were formed, one of which was identified by fast atom bombardment mass spectrometry as S-(2-phenyl)ethylglutathione. The glutathione thiyl radical intermediate was observed by spin trapping with DMPO. These results support the proposed free radical-mediated formation of styrene-glutathione conjugates by peroxidase enzymes.     

Modification of proteins by active oxygen and their degradation

A detailed analysis of literary data concerning the oxidative modification of proteins by active oxygen species was carried out. It was shown that intermediate products of molecular oxygen reduction, e.g., superoxide anion radical, hydrogen peroxide and hydroxyl radical, can induce the inactivation of enzymes in vitro as a result of oxidative modification of certain amino acid residues necessary for the maintenance of native properties of the enzyme. In some cases modification of enzymes results in their degradation by proteolytic enzymes. Besides, some enzymes catalyzing the interconversions of active oxygen species (catalase superoxide dismutase, cytochrome P-450) are also inactivated in the course of catalysis under the oxidative action of active oxygen species. It was assumed that the oxidative modification of proteins appears to be one of the mechanisms which control their degradation in the cell. The hydroxyl radical oxidizing the amino acid residues located in the vicinity of the site of its synthesis is a direct modifying species. The superoxide anion radical and hydrogen peroxide are hydroxyl radical precursors and are responsible for the transport of oxidizing equivalents in the cell.     

Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme

During the deamination of S-2-aminopropanol by the AdoCbl-dependent ethanolamine ammonia-lyase of Clostridia sp., a catalytic intermediate accumulates whose active site contains two paramagnetic species: cob(II)alamin and a free radical derived from the substrate molecule. Spin-echo spectroscopy has revealed that the unpaired electron on the substrate-derived radical is delocalized over a nitrogen atom that from its quadrupole splittings is probably a component of a secondary amide group. Experiments with 15N- and deuterium-labeled propanolamine gave no evidence of an interaction between this unpaired electron and the nitrogen originally attached to the substrate molecule. These results strongly suggest that the substrate-derived radical in this intermediate has already lost its nitrogen, and that this radical is stabilized by delocalization of the unpaired electron onto a nitrogen most likely situated in one of the peptide bonds of the enzyme backbone.     

Electron spin resonance studies on the lipoxygenase reaction by spin trapping and spin labelling methods.

The rate of oxygenation and that of trapping linoleic acid free radicals in the lipoxygenase [EC 1.13.11.12] reaction were measured in the presence of linoleic acid, oxygen, and nitrosobenzene at various concentrations, with a Clark oxygen electrode and ESR spectroscopy. The results were interpreted under the assumption that the free radical of linoleic acid, an intermediate of the lipoxygenase reaction, reacts competitively with oxygen or nitrosobenzene. The oxidation of the iron in the active site of lipoxygenase caused by the spin label reagent, 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxyl, was also observed by ESR- and fluorescence-spectroscopy.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Evaluation of classical NSAIDs and COX-2 selective inhibitors on purified ovine enzymes and human whole blood.

Cyclooxygenase is the key enzyme in the biosynthesis of prostanoids, biologically active substances involved in several physiological processes and also in pathological conditions such as inflammation. It has been well known for 10 years that this enzyme exists under two forms: a constitutive (COX-1) and an inducible form (COX-2). Both enzymes are sensitive to inhibition by conventional non-steroidal anti-inflammatory drugs (NSAIDs). Observations were made that COX-1 was mainly involved in homeostatic processes, while the COX-2 expression was associated with pathological conditions leading to the development of COX-2 selective inhibitors. Several methods have been reported for the evaluation of the COX-1 and COX-2 inhibitory potency and selectivity of conventional or COX-2 selective NSAIDs. In this study, we evaluated the COXs inhibitory profile of both conventional NSAIDs and COX-2 selective inhibitors using two different in vitro methods: the first test was performed using purified enzymes while the second method consisted of a whole blood assay. The results obtained with reference drugs in these two assays will be discussed and compared in this article.     

Selective COX-2 inhibitors. Part 1: synthesis and biological evaluation of phenylazobenzenesulfonamides

A series of phenylazobenzenesulfonamide derivatives were designed and synthesized for the evaluation as selective cyclooxygenase-2 (COX-2) inhibitors in a cellular assay using human whole blood (HWB) and an enzymatic assay using purified ovine enzymes. Extensive structure-activity relationships (SAR) were studied within this series, and several of selective COX-2 inhibitors have been identified. Among them, compound 8, 4-(4-amino-2-methylsulfanyl-phenylazo)benzenesulfonamide, showed a potent inhibitory activity to the cyclooxygenase enzymes (IC(50)'s for COX-1: 23.28 microM; COX-2: 2.04 microM), being active but less COX-2 selective than celecoxib.     

QSAR analysis of meclofenamic acid analogues as selective COX-2 inhibitors

The use of quantitative structure-activity relationships, since its advent, has become increasingly helpful in understanding many aspects of biochemical interactions in drug research. This approach was utilized to explain the relationship of structure with biological activity of selective COX-2 inhibitors. The enormity of the COX-2 discovery is reflected in the unprecedented speed at which research laboratories have sought to validate its clinical implications. Presented herein is a series of 21 derivatives of meclofenamic acid with selective COX-2 inhibitory activity. Several statistically significant regression expressions were obtained for both COX-1 and COX-2 inhibition using sequential multiple linear regression analysis method. Two of these models were selected and validated further, which revealed the importance of Kier molecular flexibility index for COX-2 inhibitory activity and the number of hydrogen bond donor atoms for COX-1 inhibitory activity. Additionally, linear correlation of molecular flexibility with COX-1 and COX-2 inhibitory activities revealed that flexibility of molecules at COX-2 active site can improve the selectivity of COX-2 inhibitors.     

Brownian dynamics simulations of aldolase binding glyceraldehyde 3-phosphate dehydrogenase and the possibility of substrate channeling

Brownian dynamics (BD) simulations test for channeling of the substrate, glyceraldehyde 3-phosphate (GAP), as it passes between the enzymes fructose-1,6-bisphosphate aldolase (aldolase) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). First, BD simulations determined the favorable complexes between aldolase and GAPDH; two adjacent subunits of GAPDH form salt bridges with two subunits of aldolase. These intermolecular contacts provide a strong electrostatic interaction between the enzymes. Second, BD simulates GAP moving out of the active site of the A or D aldolase subunit and entering any of the four active sites of GAPDH. The efficiency of transfer is determined as the relative number of BD trajectories that reached any active site of GAPDH. The distribution functions of the transfer time were calculated based on the duration of successful trajectories. BD simulations of the GAP binding from solution to aldolase/GAPDH complex were compared to the channeling simulations. The efficiency of transfer of GAP within an aldolase/GAPDH complex was 2 to 3% compared to 1.3% when GAP was binding to GAPDH from solution. There is a preference for GAP channeling between aldolase and GAPDH when compared to binding from solution. However, this preference is not large enough to be considered as a theoretical proof of channeling between these proteins.     

Synthesis,COX-1/2 inhibition activities and molecular docking study of isothiazolopyridine derivatives

One of the main challenges for nowadays medicine is drugs selectivity. In COX-1 and COX-2, the active sites are composed of the same group of amino acids with the exception of the only one residue in position 523, in COX-1 is an isoleucine, while in COX-2 is a valine. Here, we presented a series of isothiazolopyridine/benzisothiazole derivatives substituted differently into an isothiazole ring, which were synthesized and investigated for their potencies to inhibit COX-1 and COX-2 enzymes by colorimetric inhibitor screening assay. All the tested compounds inhibited the activity of COX-1, the effect on COX-2 activity was differential. The mode of binding was characterized by a molecular docking study. Comparing biological activity of the investigated compounds, it was observed that compounds sharing the most similar position to flurbiprofen and meloxicam, representing the two main enzyme subdomains, achieved higher biological activity than others. It is directly related to the fit to the enzyme’s active site, which prevents too early dissociation of the compounds.     

Fluorescence quenching analysis of the association and dissociation of a diarylheterocycle to cyclooxygenase-1 and cyclooxygenase-2: dynamic basis of cyclooxygenase-2 selectivity

Cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) are the enzymes responsible for the biosynthesis of the precursor to the biologically active prostaglandins, prostacyclin, and thromboxane and are the molecular targets for nonsteroidal antiinflammatory drugs (NSAIDs). Selective COX-2 inhibitors are antiinflammatory and analgesic but lack gastrointestinal toxicity, an undesirable side effect attributed to COX-1 inhibition. Crystallographic analysis of selective COX inhibitors complexed with either isoform provides some information about the molecular determinants of selectivity but does not provide information about the dynamics of inhibitor association/dissociation. We employed rapid-mixing techniques and fluorescence quenching to monitor the association and dissociation of a selective COX-2 inhibitor to COX-1 or COX-2. The association of the fluorescent diaryloxazole, SC299, with both enzymes occurs in a time-dependent fashion. Its binding to COX-2 occurs in three kinetically distinct steps whereas its binding to COX-1 occurs in two steps. In contrast to the relatively rapid association of SC299 with both enzymes, its dissociation from COX-2 is quite slow and occurs over several hours whereas the dissociation from COX-1 is complete in less than 1 min. The selectivity of SC299 as a COX-2 inhibitor correlates to its relative rates of dissociation from the two COX isoforms. A model is proposed for diarylheterocycle binding to COX's that integrates these kinetic data with available structural information.     

Effects of COX-2 inhibitors on ROS produced by Chlamydia pneumoniae-primed human promonocytic cells (THP-1)

Chronic inflammation through foam cells and macrophages is important in atherosclerosis development, and can be considered as therapeutic targets. Cyclooxygenase and NADPH-oxidase were expressed within atherosclerotic lesions. Reactive oxygen species produced by NADPH oxidase were found to trigger the cyclooxygenase-2 expression. The effects of preferential COX-2 inhibitors on ROS produced by Chlamydia-primed human monocytes (THP-1 cells) were evaluated by fluorescence, chemiluminescence, oxymetry, and EPR spin trapping. Fluorescence assays showed an increased production of ROS with Chlamydia versus cells primed by 10(-8)M PMA. COX-2 inhibitors inhibited in a dose-dependent manner the luminol-enhanced CL while ibuprofen and diclofenac increased the chemiluminescence response. By EPR spin trapping, COX-2 inhibitors, ibuprofen, and diclofenac, exhibited a dose-dependent inhibiting effect (10 and 100muM) on the EPR signal appearance. Our cell model combining EPR, chemiluminescence, and oxymetry appeared relevant to study the modulating effects of preferential COX-2 inhibitors on the cell oxidant activity and chronic inflammatory diseases.     

EPR spin trapping of an oxalate-derived free radical in the oxalate decarboxylase reaction

EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly (13)C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution.     

Synthesis and evaluation of fluorobenzoylated di- and tripeptides as inhibitors of cyclooxygenase-2 (COX-2)

A series of fluorobenzoylated di- and tripeptides as potential leads for the development of molecular probes for imaging of COX-2 expression was prepared according to standard Fmoc-based solid-phase peptide synthesis. All peptides were assessed for their COX-2 inhibitory potency and selectivity profile in a fluorescence-based COX binding assay. Within the series of 15 peptides tested, cysteine-containing peptides numbered 7, 8, 11 and 12, respectively, were the most potent COX-2 inhibitors possessing IC(50) values ranging from 5 to 85 μM. Fluorobenzoylated tripeptides 7 and 8 displayed some COX-2 selectivity (COX-2 selectivity index 2.1 and 1.6), whereas fluorobenzoylated dipeptides 11 and 12 were shown not to be COX-2 selective. Fluorbenzoylated tripeptide FB-Phe-Cys-Ser-OH was further used in molecular modeling docking studies to determine the binding mode within the active site of the COX-2 enzyme.     

Brownian dynamic study of an enzyme metabolon in the TCA cycle: Substrate kinetics and channeling

Malate dehydrogenase (MDH) and citrate synthase (CS) are two pacemaking enzymes involved in the tricarboxylic acid (TCA) cycle. Oxaloacetate (OAA) molecules are the intermediate substrates that are transferred from the MDH to CS to carry out sequential catalysis. It is known that, to achieve a high flux of intermediate transport and reduce the probability of substrate leaking, a MDH‐CS metabolon forms to enhance the OAA substrate channeling. In this study, we aim to understand the OAA channeling within possible MDH‐CS metabolons that have different structural orientations in their complexes. Three MDH‐CS metabolons from native bovine, wild‐type porcine, and recombinant sources, published in recent work, were selected to calculate OAA transfer efficiency by Brownian dynamics (BD) simulations and to study, through electrostatic potential calculations, a possible role of charges that drive the substrate channeling. Our results show that an electrostatic channel is formed in the metabolons of native bovine and recombinant porcine enzymes, which guides the oppositely charged OAA molecules passing through the channel and enhances the transfer efficiency. However, the channeling probability in a suggested wild‐type porcine metabolon conformation is reduced due to an extended diffusion length between the MDH and CS active sites, implying that the corresponding arrangements of MDH and CS result in the decrease of electrostatic steering between substrates and protein surface and then reduce the substrate transfer efficiency from one active site to another.     

Metabolic activation of 1,2-dibromoethane to a free radical intermediate by rat liver microsomes and isolated hepatocytes

A one-electron reductive metabolism of 1,2-dibromoethane (DBE) is described that gives rise to a free radical intermediate, which can be stabilized by a spin trapping agent and detected by electron spin resonance spectroscopy. Using rat liver microsomes or isolated hepatocytes from phenobarbitone pretreated animals, under hypoxic conditions, it has been possible to trap a free radical intermediate and identify it by using ¹³C-DBE. Inhibition experiments have demonstrated that the site of activation is the microsomal drug metabolizing system.     

Localization of a catalytic intermediate bound to the FeMo-cofactor of nitrogenase

Nitrogenase catalyzes the biological reduction of N(2) to ammonia (nitrogen fixation) as well as the reduction of a number of alternative substrates, including acetylene (HC identical with CH) to ethylene (H2C=CH2). It is known that the metallocluster FeMo-cofactor located within the nitrogenase MoFe protein component provides the site of substrate reduction, but the exact site where substrates bind and are reduced on the FeMo-cofactor remains unknown. We have recently shown that the alpha-70 residue of the MoFe protein plays a significant role in defining substrate access to the active site; alpha-70 approaches one face of the FeMo-cofactor, and when valine is substituted by alanine at this position, the substituted nitrogenase is able to accommodate a reduction of the larger alkyne propargyl alcohol (HC identical with CCH(2)OH, propargyl-OH). During this reduction, a substrate-derived intermediate can be trapped on the FeMo-cofactor resulting in an S = 1/2 spin system with a novel electron paramagnetic resonance spectrum. In the present work, trapping of the propargyl-OH-derived or propargyl amine (HC identical with CCH(2)NH(2), propargyl-NH(2))-derived intermediates is shown to be dependent on pH and the presence of histidine at position alpha-195. It is concluded that these catalytic intermediates are stabilized and thereby trapped by H-bonding interactions between either the-OH group or the-NH(3)(+)group and the imidazole epsilon-NH of alpha-195(His). Thus, for the first time it is possible to establish the location of a bound substrate-derived intermediate on the FeMo-cofactor. Refinement of the binding mode and site was accomplished by the use of density functional and force field calculations pointing to an eta(2) coordination at Fe-6 of the FeMo-cofactor.     

Toward the understanding of the molecular basis for the inhibition of COX-1 and COX-2 by phenolic compounds present in Uruguayan propolis and grape pomace

Propolis and grape pomace have significant amounts of phenols which can take part in anti-inflammatory mechanisms. As the cyclooxygenases 1 and 2 (COX-1 and COX-2) are involved in said mechanisms, the possibility for a selective inhibition of COX-2 was analyzed in vitro and in silico. Propolis and grape pomace from Uruguayan species were collected, extracted in hydroalcoholic mixture and analyzed. Based on phenols previously identified, and taking as reference the crystallographic structures of COX-1 and COX-2 in complex with the commercial drug Celecoxib, a molecular docking procedure was devised to adjust 123 phenolic molecular models at the enzyme-binding sites. The most important results of this work are that the extracts have an overall inhibition activity very similar in COX-1 and COX-2, i.e. they do not possess selective inhibition activity for COX-2. Nevertheless, 10 compounds of the phenolic database turned out to be more selective and 94 phenols resulted with similar selectivity than Celecoxib, an outcome that accounts for the overall experimental inhibition measures. Binding site environment observations showed increased polarity in COX-2 as compared with COX-1, suggesting that polarity is the key for selectivity. Accordingly, the screening of molecular contacts pointed to the residues: Arg106, Gln178, Leu338, Ser339, Tyr341, Tyr371, Arg499, Ala502, Val509, and Ser516, which would explain, at the atomic level, the anti-inflammatory effect of the phenolic compounds. Among them, Gln178 and Arg499 appear to be essential for the selective inhibition of COX-2.