Structural Basis for Certain Naturally Occurring Bioflavonoids to Function as Reducing Co-Substrates of Cyclooxygenase I and II

Recent studies showed that some of the dietary bioflavonoids can strongly stimulate the catalytic activity of cyclooxygenase (COX) I and II in vitro and in vivo, presumably by facilitating enzyme re-activation. In this study, we sought to understand the structural basis of COX activation by these dietary compounds.A combination of molecular modeling studies, biochemical analysis and site-directed mutagenesis assay was used as research tools. Three-dimensional quantitative structure-activity relationship analysis (QSAR/CoMFA) predicted that the ability of bioflavonoids to activate COX I and II depends heavily on their B-ring structure, a moiety known to be associated with strong antioxidant ability. Using the homology modeling and docking approaches, we identified the peroxidase active site of COX I and II as the binding site for bioflavonoids. Upon binding to this site, bioflavonoid can directly interact with hematin of the COX enzyme and facilitate the electron transfer from bioflavonoid to hematin. The docking results were verified by biochemical analysis, which reveals that when the cyclooxygenase activity of COXs is inhibited by covalent modification, myricetin can still stimulate the conversion of PGG(2) to PGE(2), a reaction selectively catalyzed by the peroxidase activity. Using the site-directed mutagenesis analysis, we confirmed that Q189 at the peroxidase site of COX II is essential for bioflavonoids to bind and re-activate its catalytic activity.These findings provide the structural basis for bioflavonoids to function as high-affinity reducing co-substrates of COXs through binding to the peroxidase active site, facilitating electron transfer and enzyme re-activation

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

The computed binding energy values (Δ<i>E</i>_binding) for the molecular docking study for the binding of myricetin, quercetin, chrysin or flavone with human COX I or COX II.

<p>The ligand-enzyme interaction energy value (Δ<i>E</i>_binding) was calculated using the following equation: Δ<i>E</i>_binding = <i>E</i>_complex−(<i>E</i>_COX+<i>E</i>_ligand), where <i>E</i>_complex was the potential energy for the complex of COX bound with the ligand, <i>E</i>_COX was the potential energy of the enzyme alone, and <i>E</i>_ligand was the potential energy for the ligand alone.</p

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

Chemical structures of the bioflavonoids used in this study.

<p>The structure of flavone is enlarged to show the numbering of different carbon positions.</p

Schematic depiction of the catalysis and inactivation mechanism of COX enzymes and their interaction with bioflavonoids.

<p>PPIX is for protoporphorin IX. Quercetin structure is shown as a representative bioactive bioflavonoid. Events in the peroxidase cycle are labeled with numbers to denote the sequence of occurrence.</p

The 3-D QSAR/CoMFA analysis showing the correlation between the experimentally-determined COX-stimulating activity values and the predicted values.

<p>Nine representative bioflavonoids plus flavone were used in the analysis. The experimental values were based on measuring PGE₂ production, which was determined in our previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012316#pone.0012316-Bai1" target="_blank">[15]</a>. Statistical parameters (<i>q</i>², <i>r</i>², PC, SEE, and F) for the CoMFA models of COX I and II are also listed in the figure.</p

Myricetin stimulates the catalytic activity of COX I and II (with or without aspirin pretreatment) when [¹⁴C]AA or PGG₂ is used as substrate.

<p>The incubation mixtures consisted of 20 µM [¹⁴C]AA (0.2 µCi) or 10 µM PGG₂ as substrate, COX I or COX II as enzyme (0.5 or 0.97 µg/mL, respectively), 10 mM EDTA, 1 mM reduced glutathione, 1 µM hematin, and myricetin in 200 µL Tris-HCl buffer (100 mM, pH 7.4). The reaction was incubated at 37°C for 5 min and terminated by adding 15 µL of 0.5 N HCl to each test tube. Ethyl acetate (600 µL) was added immediately for extraction. The dried extracts were re-dissolved in acetonitrile or EIA buffer (Cayman Co. Michigan, USA), and the metabolites were analyzed using HPLC (with radioactivity detection) when [¹⁴C]AA was used as substrate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012316#pone.0012316-Bai1" target="_blank">[15]</a> or using an EIA kit when PGG₂ was used as substrate. Note that in this experiment, the COX I and II enzymes with or without aspirin pretreatment were both tested. For aspirin pretreatment, enzymes were pre-incubated with aspirin at 0.5 mM for COX I or 5 mM for COX II for 30 min at room temperature and then were immediately used as the enzyme source in the assay.</p