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

Abstract 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.

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Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level Functional and modelling studies

Thrombosis and Haemostasis ◽

10.1160/th09-01-0057 ◽

2009 ◽

Vol 102 (08) ◽

pp. 336-346 ◽

Cited By ~ 35

Author(s):

Marilena Crescente ◽

Gisela Jessen ◽

Stefania Momi ◽

Hans-Dieter Höltje ◽

Paolo Gresele ◽

...

Keyword(s):

Salicylic Acid ◽

Platelet Aggregation ◽

Gallic Acid ◽

Molecular Modelling ◽

In Silico Docking ◽

Cyclooxygenase 1 ◽

Modelling Studies ◽

Cox 1

SummaryWhile resveratrol and quercetin possess antiplatelet activity, little is known on the effect of gallic acid on platelets.We studied the interactions of these three different polyphenols among themselves and with aspirin, at the level of platelet cyclooxygenase-1 (COX-1). Both functional (in vitro and in vivo) and molecular modelling approaches were used. All three polyphenols showed comparable antioxidant activity (arachidonic acid [AA]-induced intraplatelet ROS production); however, resveratrol and quercetin, but not gallic acid, inhibited AA-induced platelet aggregation. Gallic acid, similarly to salicylic acid, the major aspirin metabolite, prevented inhibition of AA-induced platelet function by aspirin but, at variance with salicylic acid, also prevented inhibition by the other two polyphenols. Molecular modelling studies, performed by in silico docking the polyphenols into the crystal structure of COX-1, suggested that all compounds form stable complexes into the COX-1 channel, with slightly different but functionally relevant interaction geometries. Experiments in mice showed that gallic acid administered before aspirin, resveratrol or quercetin fully prevented their inhibitory effect on serum TxB2. Finally, a mixture of resveratrol, quercetin and gallic acid, at relative concentrations similar to those contained in most red wines, did not inhibit platelet aggregation, but potentiated sub-inhibitory concentrations of aspirin. Gallic acid interactions with other polyphenols or aspirin at the level of platelet COX-1 might partly explain the complex,and possibly contrasting, effects of wine and other components of the Mediterranean diet on platelets and on the pharmacologic effect of lowdose aspirin.

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Cyclooxygenase-1 haplotype C50T/A-842G does not affect platelet response to aspirin

Thrombosis and Haemostasis ◽

10.1160/th08-11-0753 ◽

2009 ◽

Vol 101 (04) ◽

pp. 687-690 ◽

Cited By ~ 25

Author(s):

Caterina Pettinella ◽

Mario Romano ◽

Liborio Stuppia ◽

Francesca Santilli ◽

Rossella Liani ◽

...

Keyword(s):

Low Dose ◽

Ex Vivo ◽

Aspirin Resistance ◽

Platelet Response ◽

Dose Aspirin ◽

Cyclooxygenase 1 ◽

Low Dose Aspirin ◽

Induced Aggregation ◽

Cox 1

SummaryCOX-1 polymorphism C50T, in complete linkage disequilibrium with the other polymorphism A-842G, has been depicted as a determinant of pharmacological response to aspirin treatment. Whether these polymorphisms exert an effect on response to aspirin both in vitro and ex vivo is still controversial. We geno-typed a population of 148 healthy individuals for the C50T/A-842G haplotype. Thirty of them underwent low-dose aspirin (100 mg daily) treatment for four weeks and were followed up for seven days after withdrawal. In this subgroup, we evaluated the thromboxane-dependence of biochemical and functional indexes used to monitor the antiplatelet effect of low-dose aspirin. Among the 148 subjects studied, 10 were heterozygous for the C50T/A-842G haplotype (6.7%) and only one was homozygous for the 50T/-842G haplotype (0.67%). In the group on low-dose aspirin, serum thromboxane (TX) B2 as well as urinary 11-dehydro-TXB2 and arachidonic acid (AA)-induced aggregation were similarly suppressed in carriers and non-carriers of the 50T/-842G haplotype, with an increase until basal levels of all the parameters within seven days after withdrawal. We found no relationship between the 50T/-842G haplotype and the so-called phenomenon of aspirin resistance. Platelet cyclooxygenase activity, as reflected by serum TXB2, was uniformly and persistently suppressed by low-dose aspirin in both carriers and non carriers of these polymorphisms.

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A semi-rational design strategy of directed evolution combined with chemical synthesis of DNA sequences

Biological Chemistry ◽

10.1515/bc.2007.153 ◽

2007 ◽

Vol 388 (12) ◽

pp. 1291-1300 ◽

Cited By ~ 10

Author(s):

Ai-Sheng Xiong ◽

Ri-He Peng ◽

Jing Zhuang ◽

Jin-Ge Liu ◽

Feng Gao ◽

...

Keyword(s):

Directed Evolution ◽

Dna Sequences ◽

Active Sites ◽

Rational Design ◽

Specific Activity ◽

Dna Shuffling ◽

Gene Encoding ◽

Library Construction ◽

Key Sites

Abstract Directed evolution in vitro is a powerful molecular tool for the creation of new biological phenotypes. It is unclear whether it is more efficient to mutate an enzyme randomly or to mutate just the active sites or key sites. In this study, the strategy of a semi-rational design of directed evolution combined with whole sequence and sites was developed. The 1553 bp gene encoding the thermostable β-galactosidase of Pyrococcus woesei was chemically synthesized and optimized for G+C content and mRNA secondary structures. The synthesized gene product was used as a template or as a wild-type control. On the basis of the first round of DNA shuffling, library construction and screening, one mutant of YH6754 was isolated with higher activity. Eight potential key sites were deduced from the sequence of the shuffled gene, and 16 degenerate oligonucleotides were designed according to those eight amino acids. Two variants of YG6765 and YG8252 were screened in the second part of DNA shuffling, library construction and screening. For comparison, one mutant of YH8757 was screened through the same routine rounds of directed evolution with YH6754 as template. The purified β-galactosidase from YH8757 exhibited a lower specific activity at 25°C than those purified from mutated YG6755 and YG8252.

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A Novel, Nonpeptidic, Orally Active Bivalent Inhibitor of Human β-Tryptase

Pharmacology ◽

10.1159/000492078 ◽

2018 ◽

Vol 102 (5-6) ◽

pp. 233-243 ◽

Cited By ~ 2

Author(s):

Sarah F. Giardina ◽

Douglas S. Werner ◽

Maneesh Pingle ◽

Donald E. Bergstrom ◽

Lee D. Arnold ◽

...

Keyword(s):

Active Sites ◽

Rational Design ◽

Therapeutic Potential ◽

X Ray Crystallography ◽

Inflammatory Stimuli ◽

Invaluable Tool ◽

Xenograft Models ◽

Orally Bioavailable

β-Tryptase is released from mast cells upon degranulation in response to allergic and inflammatory stimuli. Human tryptase is a homotetrameric serine protease with 4 identical active sites directed toward a central pore. These active sites present an optimized scenario for the rational design of bivalent inhibitors, which bridge 2 adjacent active sites. Using (3-[1-acylpiperidin-4-yl]phenyl)methanamine as the pharmacophoric core and a disiloxane linker to span 2 active sites we have successfully produced a novel bivalent tryptase inhibitor, compound 1a, with a comparable profile to previously described inhibitors. Pharmacological properties of compound 1a were studied in a range of in vitro enzymic and cellular screening assays, and in vivo xenograft models. This non-peptide inhibitor of tryptase demonstrated superior activity (IC50 at 100 pmol/L tryptase = 1.82 nmol/L) compared to monomeric modes of inhibition. X-ray crystallography validated the dimeric mechanism of inhibition, and 1a demonstrated good oral bioavailability and efficacy in HMC-1 xenograft models. Furthermore, compound 1a demonstrated extremely slow off rates and high selectivity against-related proteases. This highly potent, orally bioavailable and selective inhibitor of human tryptase will be an invaluable tool in future studies to explore the therapeutic potential of attenuating the activity of this elusive target.

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In vitro Anti-inflammatory Activity of Ligustrum vulgare Extracts and Their Analytical Characterization

Natural Product Communications ◽

10.1177/1934578x1300801102 ◽

2013 ◽

Vol 8 (11) ◽

pp. 1934578X1300801 ◽

Cited By ~ 1

Author(s):

Anna Macková ◽

Pavel Mučaji ◽

Ute Widowitz ◽

Rudolf Bauer

Keyword(s):

Inhibitory Activity ◽

Inhibitory Effect ◽

Neutrophil Granulocytes ◽

Cyclooxygenase 1 ◽

Ligustrum Vulgare ◽

China And Japan ◽

Cox 2 ◽

Anti Inflammatory ◽

Cox 1

Interest in the anti-inflammatory effects of Ligustrum vulgare L., which has been used traditionally in China and Japan prompted us to determine anti-inflammatory effects of the plant's compounds in leukocytes. The leaves of L. vulgare were used to prepare a decoction which was successively extracted with organic solvents (dichloromethane (DCM), n-butanol, ethyl acetate) using liquid-liquid partition. Extracts were tested for inhibition of LTB4, resp. PGE2 biosynthesis. Each extract was evaluated for its in vitro cyclooxygenase-1/2 (COX-1/2) inhibitory activity using assays with purified COX-1 and COX-2 enzymes, as well as for their LTB4 formation inhibitory activity using an assay with activated human neutrophil granulocytes. All extracts reported inhibitory actions against COXs in comparison with the synthetic inhibitors NS-398 (IC50 = 2.6 μM) and indomethacin (IC50 = 0.9 μM). The dichloromethane extract of privet leaves showed a considerable inhibitory effect against COX-1 and COX-2 enzyme activity. The DCM extract revealed 2.7 times higher inhibitory activity against LTB4 formation in comparison with the known specific LT inhibitor zileuton (IC50 = 5.0 μM). Additionally, oleuropein and echinacoside were detected by HPLC-DAD and LC-MS in the Ligustrum vulgare leaves. Both compounds exhibited weak inhibitory activity on cyclooxygenases and leukotriene formation.

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Dual COX and 5-LOX inhibition by clerodane diterpenes from seeds of Polyalthia longifolia (Sonn.) Thwaites

Scientific Reports ◽

10.1038/s41598-020-72840-8 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Ha Thi Nguyen ◽

Thien-Y. Vu ◽

Vishala Chandi ◽

Haritha Polimati ◽

Vinay Bharadwaj Tatipamula

Keyword(s):

Underlying Mechanism ◽

Study Results ◽

Clerodane Diterpenes ◽

In Silico Study ◽

Dual Inhibitors ◽

Cox 2 ◽

Lox Inhibitors ◽

Polyalthia Longifolia ◽

Cox 1

Abstract Natural metabolites with their specific bioactivities are being considered as a potential source of materials for pharmacological studies. In this study, we successfully isolated and identified five known clerodane diterpenes, namely 16-oxo-cleroda-3,13(14)E-dien-15-oic acid (1), 16-hydroxy-cleroda-3,13-dien-15-oic acid (2), 16-hydroxy-cleroda-4(18),13-dien-16,15-olide (3), 3α,16α-dihydroxy-cleroda-4(18),13(14)Z-dien-15,16-olide (4), and 16α-hydroxy-cleroda-3,13(14)Z-dien-15,16-olide (5) from the methanolic extract of seeds of Polyalthia longifolia. Initially, all the isolated metabolites were investigated for COX-1, COX-2, and 5-LOX inhibitory activities using the standard inhibitory kits. Of which, compounds 3, 4, and 5 exhibited to be potent COX-1, COX-2, and 5-LOX inhibitors with the IC50 values similar or lower to those of the reference drugs. To understand the underlying mechanism, these compounds were subjected to molecular docking on COX-1, COX-2, and 5-LOX proteins. Interestingly, the in silico study results were in high accordance with in vitro studies where compounds 3, 4, and 5 hits assumed interactions and binding pattern comparable to that of reference drugs (indomethacin and diclofenac), as a co-crystallized ligand explaining their remarkable dual (COX/LOX) inhibitor actions. Taken together, our findings demonstrated that compounds 3, 4, and 5 functioned as dual inhibitors of COX/5-LOX and can contribute to the development of novel, more effective anti-inflammatory drugs with minimal side-effects.

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In-vitro test system for the evaluation of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) inhibitors based on a single HPLC run with UV detection using bovine aortic coronary endothelial cells (BAECs)

Inflammation Research ◽

10.1007/s000110050752 ◽

2001 ◽

Vol 50 (5) ◽

pp. 262-269 ◽

Cited By ~ 17

Author(s):

G. Dannhardt ◽

H. Ulbrich

Keyword(s):

Endothelial Cells ◽

Test System ◽

In Vitro Test ◽

Cyclooxygenase 1 ◽

Vitro Test ◽

Cox 2 ◽

Coronary Endothelial Cells ◽

Cox 2 Inhibitors ◽

Cox 1

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In vivo aspirin supplementation inhibits nitric oxide consumption by human platelets

Blood ◽

10.1182/blood-2005-02-0664 ◽

2005 ◽

Vol 106 (8) ◽

pp. 2737-2743 ◽

Cited By ~ 15

Author(s):

P. Claire Williams ◽

Marcus J. Coffey ◽

Barbara Coles ◽

Stephanie Sanchez ◽

Jason D. Morrow ◽

...

Keyword(s):

Nitric Oxide ◽

Human Platelets ◽

Controlled Study ◽

Double Blind ◽

Beneficial Effects ◽

Cyclooxygenase 1 ◽

No Consumption ◽

Cox 1

AbstractAntiplatelet therapies improve endothelial function in atherosclerosis, suggesting that platelets regulate vascular nitric oxide (NO) bioactivity in vivo. Herein, washed platelets consumed NO on activation in an aspirin-sensitive manner, and aspirin enhanced platelet NO responses in vitro. To examine whether in vivo aspirin can inhibit platelet NO consumption, a double-blind placebo-controlled study was conducted. After a 2-week nonsteroidal anti-inflammatory drug (NSAID)–free period, healthy men were randomly assigned and administered aspirin (75 mg/d orally) or identical placebo for 14 days, then crossed over to the opposite arm. Following in vivo aspirin, NO consumption by platelets was inhibited 91%. Rate of onset and recovery following aspirin withdrawal was consistent with cyclooxygenase 1 (COX-1) inhibition. In a small substudy, NO consumption by platelets from postmenopausal women was faster in hypercholesterolemics and less sensitive to aspirin (ie, 39% versus 76% inhibition for hypercholesterolemics or normocholesterolemics, respectively). However, 150 mg aspirin/day increased inhibition of NO consumption by platelets of hypercholesterolemics to 80%. Comparisons of platelet COX-1 or -2 expression and urinary 11-dehydro-thromboxane B2 excretion suggested that aspirin was less able to block platelet activation in vivo in hypercholesterolemia. In conclusion, aspirin inhibits NO consumption by platelets from healthy subjects, but its beneficial effects on NO bioactivity may be compromised in some hypercholesterolemic patients.

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

Molecules ◽

10.3390/molecules27010219 ◽

2021 ◽

Vol 27 (1) ◽

pp. 219

Author(s):

Hyoung-Woo Bai ◽

Jina Yu ◽

Yue Wang ◽

Pan Wang ◽

Baoting Zhu

Keyword(s):

Acid Metabolism ◽

Inhibitory Effect ◽

Arachidonic Acid Metabolism ◽

Microsomal Preparation ◽

Pig Kidney ◽

Cyclooxygenase 1 ◽

Kidney Microsomes ◽

Cox 2 ◽

Cox 1

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.

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