Quercetin as an inhibitor of hemoglobin-mediated lipid oxidation: Mechanisms of action and use of molecular docking.

The antioxidant effect of quercetin on hemoglobin(Hb)-mediated lipid oxidation and the mechanisms involved were investigated. Quercetin strongly inhibited Hb-mediated lipid oxidation in washed muscle. Quercetin showed effective hydroxyl radical scavenging ability similar to butylated hydroxytoluene (BHT). Quercetin reduced metHb resulting in formation of oxyHb. Bound quercetin decreased heme dissociation from metHb. Conversion to oxyHb and decreased heme dissociation represent routes to limit Hb-mediated lipid oxidation. Electrospray ionization mass spectrometry (ESI-MS) indicated one molecule of quercetin was covalently bound to Hb α-chain. Quercetin quinone docked 3.3 Å from the thiol of αCys(H15) but not near any other Cys residues of turkey Hb. At the docking site, hydrogen bonding between quercetin quinone and amino acids of α- and ß-chain was demonstrated. This represents a path by which quercetin became covalently bound to α-chain. Molecular docking of heme proteins to polyphenols provides a template to better understand antioxidant interactions in muscle foods.

Adropin decreases endothelial monolayer permeability after cell-free hemoglobin exposure and reduces MCP-1-induced macrophage transmigration.

BACKGROUND: Cell-free heme-containing proteins mediate endothelial injury in a variety of disease states including subarachnoid hemorrhage and sepsis by increasing endothelial permeability. Inflammatory cells are also attracted to sites of vascular injury by monocyte chemotactic protein 1 (MCP-1) and other chemokines. We have identified a novel peptide hormone, adropin, that protects against hemoglobin-induced endothelial permeability and MCP-1-induced macrophage migration. METHODS: Human microvascular endothelial cells were exposed to cell-free hemoglobin (CFH) with and without adropin treatment before measuring monolayer permeability using a FITC-dextran tracer assay. mRNA and culture media were collected for molecular studies. We also assessed the effect of adropin on macrophage movement across the endothelial monolayer using an MCP-1-induced migration assay. RESULTS: CFH exposure decreases adropin expression and increases paracellular permeability of human endothelial cells. Treating cells with synthetic adropin protects against the increased permeability observed during the natural injury progression. Cell viability was similar in all groups and Hmox1 expression was not affected by adropin treatment. MCP-1 potently induced macrophage migration across the endothelial monolayer and adropin treatment effectively reduced this phenomenon. CONCLUSIONS: Endothelial injury is a hallmark of many disease states. Our results suggest that adropin treatment could be a valuable strategy in preventing heme-mediated endothelial injury and macrophage infiltration. Further investigation of adropin therapy in animal models and human tissue specimens is needed.

Acute toxicity of cyanide in aerobic respiration: Theoretical and experimental support for murburn explanation.

The inefficiency of cyanide/HCN (CN) binding with heme proteins (under physiological regimes) is demonstrated with an assessment of thermodynamics, kinetics, and inhibition constants. The acute onset of toxicity and CN's mg/Kg LD50 (µM lethal concentration) suggests that the classical hemeFe binding-based inhibition rationale is untenable to account for the toxicity of CN. In vitro mechanistic probing of CN-mediated inhibition of hemeFe reductionist systems was explored as a murburn model for mitochondrial oxidative phosphorylation (mOxPhos). The effect of CN in haloperoxidase catalyzed chlorine moiety transfer to small organics was considered as an analogous probe for phosphate group transfer in mOxPhos. Similarly, inclusion of CN in peroxidase-catalase mediated one-electron oxidation of small organics was used to explore electron transfer outcomes in mOxPhos, leading to water formation. The free energy correlations from a Hammett study and IC50/Hill slopes analyses and comparison with ligands ( CO/ H 2 S/ N 3 - ) $\left( {\text{CO}}/{{{{\text{H}}_{2}}\text{S}}/{\text{N}_{3}^{\text{-}}}\;}\; \right)$ provide insights into the involvement of diffusible radicals and proton-equilibriums, explaining analogous outcomes in mOxPhos chemistry. Further, we demonstrate that superoxide (diffusible reactive oxygen species, DROS) enables in vitro ATP synthesis from ADP+phosphate, and show that this reaction is inhibited by CN. Therefore, practically instantaneous CN ion-radical interactions with DROS in matrix catalytically disrupt mOxPhos, explaining the acute lethal effect of CN.

Inhibition of Hemoglobin Degrading Protease Falcipain-2 as a Mechanism for Anti-Malarial Activity of Triazole-Amino Acid Hybrids.

BACKGROUND: Novel drug development against malaria parasite over old conventional antimalarial drugs is essential due to rapid and indiscriminate use of drugs, which led to the emergence of resistant strains. METHODS: In this study, previously reported triazole-amino acid hybrids (13-18) are explored against Plasmodium falciparum as antimalarial agents. Among six compounds, 15 and 18 exhibited antimalarial activity against P. falciparum with insignificant hemolytic activity and cytotoxicity towards HepG2 mammalian cells. In molecular docking studies, both compounds bind into the active site of PfFP-2 and block its accessibility to the substrate that leads to the inhibition of target protein further supported by in vitro analysis. RESULTS: Antimalarial half-maximal inhibitory concentration (IC50) of 15 and 18 compounds were found to be 9.26 µM and 20.62 µM, respectively. Blood stage specific studies showed that compounds, 15 and 18 are effective at late trophozoite stage and block egress pathway of parasites. Decreased level of free monomeric heme was found in a dose dependent manner after the treatment with compounds 15 and 18, which was further evidenced by the reduction in percent of hemoglobin hydrolysis. Compounds 15 and 18 hindered hemoglobin degradation via intra- and extracellular cysteine protease falcipain-2 (PfFP-2) inhibitory activity both in in vitro and in vivo in P. falciparum. CONCLUSION: We report antimalarial potential of triazole-amino acid hybrids and their role in the inhibition of cysteine protease PfFP-2 as its mechanistic aspect.

Cell-free hemoglobin promotes primary graft dysfunction through oxidative lung endothelial injury.

Primary graft dysfunction (PGD) is acute lung injury within 72 hours of lung transplantation. We hypothesized that cell-free hemoglobin (CFH) contributes to PGD by increasing lung microvascular permeability and tested this in patients, ex vivo human lungs, and cultured human lung microvascular endothelial cells. In a nested case control study of 40 patients with severe PGD at 72 hours and 80 matched controls without PGD, elevated preoperative CFH was independently associated with increased PGD risk (odds ratio [OR] 2.75, 95%CI, 1.23-6.16, P = 0.014). The effect of CFH on PGD was magnified by reperfusion fraction of inspired oxygen (FiO2) ≥ 0.40 (OR 3.41, P = 0.031). Isolated perfused human lungs exposed to intravascular CFH (100 mg/dl) developed increased vascular permeability as measured by lung weight (CFH 14.4% vs. control 0.65%, P = 0.047) and extravasation of Evans blue-labeled albumin dye (EBD) into the airspace (P = 0.027). CFH (1 mg/dl) also increased paracellular permeability of human pulmonary microvascular endothelial cell monolayers (hPMVECs). Hyperoxia (FiO2 = 0.95) increased human lung and hPMVEC permeability compared with normoxia (FiO2 = 0.21). Treatment with acetaminophen (15 µg/ml), a specific hemoprotein reductant, prevented CFH-dependent permeability in human lungs (P = 0.046) and hPMVECs (P = 0.037). In summary, CFH may mediate PGD through oxidative effects on microvascular permeability, which are augmented by hyperoxia and abrogated by acetaminophen.

Hydroxy methoxy benzaldehyde from Sesbania grandilfora inhibits the advanced glycation end products (AGEs)-mediated fibrillation in hemoglobin.

The present study was aimed to identify the active anti-glycation constituent from the leaves of Sesbania grandiflora. Characterization of the active constituent resulted in the identification of hydroxy methoxy benzaldehyde (HMB). The potential of HMB as anti-glycation lead was analyzed by fluorescence spectroscopy, fluorescence microscopy, scanning electron microscopy (SEM) and molecular interaction studies. Our results suggested that HMB inhibited formation of early (HbA1c) and advanced glycation end products (AGEs). The amyloid-like fibrillation in hemoglobin was also inhibited by HMB. SEM images indicated the protective effect against the formation of acanthocytes. Molecular docking studies showed that HMB was interacting with hemoglobin through hydrogen bonds with Arg141, Tyr140, and Thr137. Our findings suggest that HMB could be a better anti-glycation lead molecule towards novel AGEs inhibitors.

Interaction of Azole-Based Environmental Pollutants with the Coelomic Hemoglobin from Amphitrite ornata: A Molecular Basis for Toxicity.

The toxicities of azole pollutants that have widespread agricultural and industrial uses are either poorly understood or unknown, particularly with respect to how infaunal organisms are impacted by this class of persistent organic pollutant. To identify a molecular basis by which azole compounds may have unforeseen toxicity on marine annelids, we examine here their impact on the multifunctional dehaloperoxidase (DHP) hemoglobin from the terebellid polychaete Amphitrite ornata. Ultraviolet-visible and resonance Raman spectroscopic studies showed an increase in the six-coordinate low-spin heme population in DHP isoenzyme B upon binding of imidazole, benzotriazole, and benzimidazole (Kd values of 52, 82, and 110 µM, respectively), suggestive of their direct binding to the heme-Fe. Accordingly, atomic-resolution X-ray crystal structures, supported by computational studies, of the DHP B complexes of benzotriazole (1.14 Å), benzimidazole (1.08 Å), imidazole (1.08 Å), and indazole (1.12 Å) revealed two ligand binding motifs, one with direct ligand binding to the heme-Fe, and another in which the ligand binds in the hydrophobic distal pocket without coordinating the heme-Fe. Taken together, the results demonstrate a new mechanism by which azole pollutants can potentially disrupt hemoglobin function, thereby improving our understanding of their impact on infaunal organisms in marine and aquatic environments.

Effects of pharmacological ascorbate on hemoglobin-induced cancer cell proliferation.

The high heme content in red meat is associated with an increased risk of developing cancer. Pharmacologic concentrations of ascorbate can specifically kill a wide range of cancer cells. In this study, the impact of ascorbate at pharmacologic concentrations on hemoglobin (Hb)-modulated human hepatoma HepG2 cell survival was investigated. It was found that HepG2 cells were proliferated by Hb (5-25µM), but killed by high pharmacologic concentrations of ascorbate (2-10mM). Although ascorbate at the low pharmacologic concentration (0.5mM) alone exhibited insignificant effect on cell viability, it effectively inhibited Hb (10µM)-induced cancer cell proliferation. The mechanism of this cytotoxicity was based on the production of extracellular H2O2 and involved transition iron. The influence of ascorbate on Hb-dependent redox reactions (i.e. the oxidative stability of Hb and its cytotoxic ferryl intermediate) was further investigated to illustrate the reaction mechanism of ascorbate toxicity, where H2O2 was generated in the reaction of ascorbate with Hb. Furthermore, circular dichroism demonstrated no significant change in the secondary structure of Hb after ascorbate addition and molecular docking revealed binding modes of ascorbate with Hb. These results demonstrated that ascorbate could possess anti-cancer activity through interfering in Hb-dependent redox reactions.

Synthesis of MR-49, a deiodinated analog of tetraiodothyroacetic acid (tetrac), as a novel pro-angiogenesis modulator.

The tyrosine-based hormones 3,3',5-triiodo-l-thyronine (l-T3) and l-thyroxine (l-T4) that are produced by the thyroid gland control metabolic functions. Iodothyronine deiodinase enzymes convert l-T4 to l-T3, the form of thyroid hormone critical to genomic actions within cells and regulation of metabolism, and to reverse-l-T3, a hormone isoform that is largely inactive. We used tertiary amines in a study of deiodination based on derivatives of tetraiodothyroacetic acid (tetrac)-a naturally occurring derivative of l-T4-to mimic the action of the iodothyronine deiodinases and deiodination of the outer ring iodines. Deiodinated tetrac, MR-49, was found to be pro-angiogenic, with this activity exceeding that of l-T3 and l-T4 in a hemoglobin Matrigel® plug assay of angiogenesis. Tetrac is anti-angiogenic via several nongenomic pathways, and the present studies of MR-49 reveal the critical contribution of outer ring iodines to the angiogenic properties of thyroid hormone analogues, which may have utility as pro-angiogenic pharmaceuticals.

Pharmacological activation of lysophosphatidic acid receptors regulates erythropoiesis.

Lysophosphatidic acid (LPA), a growth factor-like phospholipid, regulates numerous physiological functions, including cell proliferation and differentiation. In a previous study, we have demonstrated that LPA activates erythropoiesis by activating the LPA 3 receptor subtype (LPA3) under erythropoietin (EPO) induction. In the present study, we applied a pharmacological approach to further elucidate the functions of LPA receptors during red blood cell (RBC) differentiation. In K562 human erythroleukemia cells, knockdown of LPA2 enhanced erythropoiesis, whereas knockdown of LPA3 inhibited RBC differentiation. In CD34(+) human hematopoietic stem cells (hHSC) and K526 cells, the LPA3 agonist 1-oleoyl-2-methyl-sn-glycero-3-phosphothionate (2S-OMPT) promoted erythropoiesis, whereas the LPA2 agonist dodecyl monophosphate (DMP) and the nonlipid specific agonist GRI977143 (GRI) suppressed this process. In zebrafish embryos, hemoglobin expression was significantly increased by 2S-OMPT treatment but was inhibited by GRI. Furthermore, GRI treatment decreased, whereas 2S-OMPT treatment increased RBC counts and amount of hemoglobin level in adult BALB/c mice. These results indicate that LPA2 and LPA3 play opposing roles during RBC differentiation. The pharmacological activation of LPA receptor subtypes represent a novel strategies for augmenting or inhibiting erythropoiesis.

Steered molecular dynamics study of inhibitor binding in the internal binding site in dehaloperoxidase-hemoglobin.

The binding free energy of 4-bromophenol (4-BP), an inhibitor that binds in the internal binding site in dehaloperoxidase-hemoglobin (DHP) was calculated using Molecular Dynamics (MD) methods combined with pulling or umbrella sampling. The effects of systematic changes in the pulling speed, pulling force constant and restraint force constant on the calculated potential of mean force (PMF) are presented in this study. The PMFs calculated using steered molecular dynamics (SMD) were validated by umbrella sampling (US) in the strongly restrained regime. A series of restraint force constants ranging from 1000 down to 5 kJ/(mol nm(2)) were used in SMD simulations. This range was validated using US, however noting that weaker restraints give rise to a broader sampling of configurations. This comparison was further tested by a pulling simulation conducted without any restraints, which was observed to have a value closest to the experimentally measured free energy for binding of 4-BP to DHP based on ultraviolet-visible (UV-vis) and resonance Raman spectroscopies. The protein-inhibitor system is well suited for fundamental study of free energy calculations because the DHP protein is relatively small and the inhibitor is quite rigid. Simulation configuration structures are compared to the X-ray crystallography structures of the binding site of 4-BP in the distal pocket above the heme.

Interaction of the chlorite-based drug WF10 and chlorite with hemoglobin, methemoglobin and ferryl hemoglobin.

The interaction of the chlorite-based drug solution WF10 with human oxyhemoglobin and oxidized hemoglobin forms was investigated monitoring the corresponding spectral changes in heme states. The chlorite component of WF10 converts oxyhemoglobin into methemoglobin with a rate of 35.4 M(-1)s(-1). Methemoglobin is also formed upon the interaction of ferryl hemoglobin and WF10/chlorite. The rate of this interconversion depends on the oxidation state of ferryl hemoglobin. This rate is 114 M(-1)s(-1), when ferryl hemoglobin was generated upon reaction of oxyhemoglobin and hydrogen peroxide. A considerable higher rate (6600 M(-1)s(-1)) is measured between the chlorite components of WF10 and ferryl hemoglobin after formation of the latter species from methemoglobin. WF10/chlorite inactivates also methemoglobin as evidenced by the continuous decrease of the Soret band and all other absorbances with a rate of 8.3 M(-1)s(-1). In all interconversions, the chlorite component of WF10 was the active principle as shown in experiments applying pure chlorite at the same concentration as in WF10. Thus, WF10 is able to diminish efficiently the yield of cytotoxic hemoglobin species that might appear after excessive hemolysis of red blood cells under pathologic situations.

Synthesis and biological evaluation of novel histone deacetylases inhibitors with nitric oxide releasing activity.

A novel series of histone deacetylases inhibitors (HDACIs) containing benzofuroxan pharmacophore as nitric oxide (NO) donor were designed based on the combination principle and 'multifunctional drugs' theory. As a novel study on embedding NO donor into the structure of HDACIs, all designed hybrid compounds, especially 19d and 24d, displayed remarkable HDACs inhibitory activity and outstanding antiproliferative activity on tumor cells. Besides, they could produce high levels of NO in HCT-116 cells; furthermore, their antiproliferative activity on HCT-116 cells could be diminished by pretreatment with hemoglobin, as the NO scavenger, in a dose-dependent manner. All in all, our designed compounds displayed great inhibitory activities and might offer a prospective avenue to discover novel anti-cancer drugs.

Complexes of dual-function hemoglobin/dehaloperoxidase with substrate 2,4,6-trichlorophenol are inhibitory and indicate binding of halophenol to compound I.

The hemoglobin of sea worm Amphitrite ornata, which for historical reasons is abbreviated as DHP for dehaloperoxidase, has two physiological functions: it binds dioxygen in the ferrous state and dehalogenates halophenols, such as 2,4,6-trichlorophenol (TCP), using hydrogen peroxide as the oxidant in the ferric state. The crystal structures of three DHP variants (Y34N, Y34N/S91G, and L100F) with TCP bound show two mutually exclusive modes of substrate binding. One of them, the internal site, is deep inside the distal pocket with the phenolic OH moiety forming a hydrogen bond to the water molecule coordinated to the heme Fe. In this complex, the distal histidine is predominantly located in the closed position and also forms a hydrogen bond to the phenolic hydroxide. The second mode of TCP binding is external, at the heme edge, with the halophenol molecule forming a lid covering the entrance to the distal cavity. The distal histidine is in the open position and forms a hydrogen bond to the OH group of TCP, which also hydrogen bonds to the hydroxyl of Tyr38. The distance between the Cl4 atom of TCP and the heme Fe is 3.9 Å (nonbonding). In both complexes, TCP molecules prevent the approach of hydrogen peroxide to the heme, indicating that the complexes are inhibitory and implying that the substrates must bind in an ordered fashion: hydrogen peroxide first and TCP second. Kinetic studies confirmed the inhibition of DHP by high concentrations of TCP. The external binding mode may resemble the interaction of TCP with Compound I, the catalytic intermediate to which halophenols bind. The measured values of the apparent Km for TCP were in the range of 0.3-0.8 mM, much lower than the concentrations required to observe TCP binding in crystals. This indicates that during catalysis TCP binds to Compound I. Mutant F21W, which likely has the internal TCP binding site blocked, has ~7% of the activity of wild-type DHP.

Role of polarity of the distal pocket in the control of inhibitor binding in dehaloperoxidase-hemoglobin.

Dehaloperoxidase (DHP A), a unique multifunctional enzyme, from the marine annelid Amphitrite ornata dehalogenates 2,4,6-tribromophenol to form 2,6-dibromo-1,4-benzoquinone. The catalytic cycle of DHP is similar to that of horseradish peroxidase (HRP), involving a high-valent ferryl heme and two single-electron transfers from the aromatic substrate to the enzyme. Like HRP, DHP has been investigated as a potential bioremediation enzyme. However, DHP fails as a bioremediation enzyme because, unlike HRP, it has an internal binding cavity on the distal side of the heme capable of accommodating p-bromophenols, which act as an inhibitor of peroxidase function. Blocking internal binding in DHP may be the key to allowing the enzyme to function effectively as a peroxidase on the full range of halogenated phenols. The distal cavity of DHP is surrounded by several hydrophobic amino acids that stabilize internal binding of the monohalogenated phenols, including a leucine residue near the back edge of the heme (L100). We have expressed the L100F, L100Q, L100T, and L100V mutants of DHP in an effort to prevent internal binding and thereby convert the inhibitors into substrates. Kinetic assays and resonance Raman indicate that the peroxidase activity of the L100T and L100F mutants is increased compared to that of native DHP in the presence of 4-bromophenol (4-BP), suggesting a reduction in the inhibitor binding constant. In addition, the X-ray crystal structure of L100F clearly indicates a reduced occupancy of the 4-BP inhibitor in the distal cavity of DHP. However, at the same time, the L100F structure reveals that steric interference alone is insufficient to exclude the inhibitor. Instead, the comparison of L100T and isosteric L100V reveals that an increase in polarity plays a decisive role in excluding the inhibitor from the distal binding pocket.

The dehaloperoxidase paradox.

The dual functions of the dehaloperoxidase-hemoglobin of Amphitrite ornata leads to a paradox. Peroxidase and hemoglobin functions require ferric and ferrous resting states, respectively. Assuming that hemoglobin function is the dominant function, the starting point for peroxidase activation would be the oxyferrous state. Activation of that state leads to the ferryl intermediate, followed by one-electron oxidation of the substrate, which results in the ferric state. Since no exogenous reductant is known, there is no return to the ferrous form or hemoglobin function. The observation that an internal binding site for 4-bromophenol leads to inhibition leads to a further paradox that the enzyme would be inhibited immediately upon activation under ambient conditions in benthic ecosystems where the inhibitor, 4-bromophenol is present in greater concentration than the substrate, 2,4,6-tribromophenol. In this review, we explore the unresolved aspects of the reaction scheme that leads to the apparent paradox. Recent data showing activation of the oxyferrous state, an extremely high reduction potential and exogenous reduction by the 2,6-dibromoquinone product present a potential resolution of the paradox. These aspects are discussed in the context of control of reactivity radical pathways and reactivity by the motion of the distal histidine, H55, which in turn is coupled to the binding of substrate and inhibitor.

Implant-induced intraperitoneal inflammatory angiogenesis is attenuated by fluvastatin.

1. Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA) inhibitors, exert anti-inflammatory, anti-oxidant and anti-angiogenic effects. These effects are associated with downregulation of pro-inflammatory/pro-angiogenic molecules and upregulation of endothelial nitric oxide synthase (e-NOS) expression/nitric oxide (NO) production. 2. Using the murine sponge model to induce chronic intraperitoneal inflammatory response, we evaluated the inflammatory components, angiogenic and NO production of the fibrovascular tissue, and their modulation by fluvastatin. 3. Our results showed that fluvastatin (0.6 and 6 mg/kg per day) inhibited haemoglobin (Hb) content 4.9±0.4 (n=15; control) vs 2.2±0.2 (n=6; fluvastatin 0.6) and 1.8±0.2 (n=6; fluvastatin 6.0) and the number of vessels in the treated group when compared with the control group. The inflammatory component, as assessed by myeloperoxidase and N-acetyl-ß-d-glucosaminidase activities and by the pro-inflammatory cytokines, tumour necrosis factor-α (TNF-α) and Monocyte chemotactic protein-1 (MCP-1)/CCL2/JE levels, was also decreased by the compound. In the treated group, inhibition of both enzyme activities was 54% and 57%, respectively. The levels of the cytokines (TNF-α and CCL2/JE) intra-implant were decreased relative to the control. In these implants, fluvastatin was also able to increase NO production, as detected with an NO-sensitive electrode. 4. The inhibitory function of fluvastatin on key components of intraperitoneal inflammatory angiogenesis shown in the present study is clearly associated with the modulatory effects of this statin on vascular endothelial growth factor, TNF-α and NO production.

High-sensitivity spectrofluorimetric determination of tiopronin based on inhibition of hemoglobin.

A highly sensitive and simple spectrofluorimetric method for the determination of tiopronin based on its inhibitory effect on the hemoglobin-catalyzed reaction of H(2)O(2) and L-tyrosine was developed. The concentration of tiopronin is linear with decreased fluorescence (ΔF) of the system under the optimal experimental conditions. The calibration graph is linear in the range 1.23 × 10(-8) to 3.06 × 10(-5) mol L(-1) with a detection limit of 6.13 × 10(-9) mol L(-1). The relative standard deviation was 4.38% for 11 determinations of 6.13 × 10(-6) mol L(-1). This method can be used for the determination of tiopronin in pharmaceuticals with satisfactory results.