Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors

A myeloperoxidase inhibitory kaempferol derivative, namely pterogynoside (1), was isolated from fruits of Pterogyne nitens, along with six known flavonols, kaempferol, afzelin, kaempferitrin, quercetin, isoquercetrin and rutin. The structures of all compounds were elucidated primarily from 1D and 2D NMR spectroscopic analyses, as well as by high resolution mass spectrometry. All flavonols were screened to identify secondary metabolites as potential myeloperoxidase (MPO) inhibitors, and at concentrations of 0.50-50 nM, quercetin (5), isoquercitrin (6) and rutin (7) exhibited strong inhibitory effects with IC(50) values of 1.22 +/- 0.01, 3.75 +/- 0.02 and 3.60 +/- 0.02, respectively. The MPO activity detected for the new derivative 1 was markedly decreased (IC(50) 10.3 +/- 0.03) when compared with known flavonols 5-7, and interestingly increased when tested against ABTS scavenging activity. (C) 2008 Published by Elsevier Ltd.

MPO Inhibitors Selected by Virtual Screening

The hemeprotein myeloperoxidase (MPO) participates in innate immune defense through its ability to generate potent microbicidal oxidants. However, these oxidants are also key mediators of the tissue damage associated with many inflammatory diseases. Thus, there is considerable interest in developing therapeutically useful MPO inhibitors. Here, we used structure-based drug design (SBDD) and ligand-based drug design (LBDD) to select for potentially new and selective MPO inhibitors. A pharmacophore model was developed based on the crystal structure of human MPO in complex with salicylhydroxamic acid (SHA), a known inhibitor of the enzyme. The pharmacophore model was used to screen the ZINC database for potential ligands, which were further filtered on the basis of their physical-chemical properties and docking score. The filtered compounds were visually inspected, and nine were purchased for experimental studies. Surprisingly, almost all of the selected compounds belonged to the aromatic hydrazide class, which had been previously described as MPO inhibitors. The compounds selected by virtual screening were shown to inhibit the chlorinating activity of MPO; the top four compounds displayed IC(50) values ranging from 1.0 to 2.8 mM. MPO inactivation by the most effective compound was shown to be irreversible. Overall, our results show that SBDD and LBDD may be useful for the rational development of new MPO inhibitors.

4 '-Aminochalcones As Novel Inhibitors of the Chlorinating Activity of Myeloperoxidase

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of myeloperoxidase-mediated hypochlorous acid production by nitroxides

Tissue damage resulting from the extracellular production of HOCl (hypochlorous acid) by the MPO (myeloperoxidase)-hydrogen peroxide-chloride system of activated phagocytes is implicated as a key event in the progression of a number of human inflammatory diseases. Consequently, there is considerable interest in the development of therapeutically useful MPO inhibitors. Nitroxides are well established antioxidant compounds of low toxicity that can attenuate oxidative damage in animal models of inflammatory disease. They are believed to exert protective effects principally by acting as superoxide dismutase mimetics or radical scavengers. However, we show here that nitroxides can also potently inhibit MPO-mediated HOCl production, with the nitroxide 4-aminoTEMPO inhibiting HOCl production by MPO and by neutrophils with IC50 values of approx. 1 and 6 μM respectively. Structure–activity relationships were determined for a range of aliphatic and aromatic nitroxides, and inhibition of oxidative damage to two biologically-important protein targets (albumin and perlecan) are demonstrated. Inhibition was shown to involve one-electron oxidation of the nitroxides by the compound I form of MPO and accumulation of compound II. Haem destruction was also observed with some nitroxides. Inhibition of neutrophil HOCl production by nitroxides was antagonized by neutrophil-derived superoxide, with this attributed to superoxide-mediated reduction of compound II. This effect was marginal with 4-aminoTEMPO, probably due to the efficient superoxide dismutase-mimetic activity of this nitroxide. Overall, these data indicate that nitroxides have considerable promise as therapeutic agents for the inhibition of MPO-mediated damage in inflammatory diseases.

Inhibition of myeloperoxidase- and neutrophil-mediated oxidant production by tetraethyl and tetramethyl nitroxides

The powerful oxidant HOCl (hypochlorous acid and its corresponding anion, −OCl) generated by the myeloperoxidase (MPO)–H2O2–Cl− system of activated leukocytes is strongly associated with multiple human inflammatory diseases; consequently there is considerable interest in inhibition of this enzyme. Nitroxides are established antioxidants of low toxicity that can attenuate oxidation in animal models, with this ascribed to superoxide dismutase or radical-scavenging activities. We have shown (M.D. Rees et al., Biochem. J. 421, 79–86, 2009) that nitroxides, including 4-amino-TEMPO (4-amino-2,2,6,6-tetramethylpiperidin-1-yloxyl radical), are potent inhibitors of HOCl formation by isolated MPO and activated neutrophils, with IC50 values of ~1 and ~6 µM respectively. The utility of tetramethyl-substituted nitroxides is, however, limited by their rapid reduction by biological reductants. The corresponding tetraethyl-substituted nitroxides have, however, been reported to be less susceptible to reduction. In this study we show that the tetraethyl species were reduced less rapidly than the tetramethyl species by both human plasma (89–99% decreased rate of reduction) and activated human neutrophils (62–75% decreased rate). The tetraethyl-substituted nitroxides retained their ability to inhibit HOCl production by MPO and activated neutrophils with IC50 values in the low-micromolar range; in some cases inhibition was enhanced compared to tetramethyl substitution. Nitroxides with rigid structures (fused oxaspiro rings) were, however, inactive. Overall, these data indicate that tetraethyl-substituted nitroxides are potent inhibitors of oxidant formation by MPO, with longer plasma and cellular half-lives compared to the tetramethyl species, potentially allowing lower doses to be employed.

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation. Here, we synthesized diapocynin and studied its effect on inhibition of gp91(phox) RNA expression. We found that diapocynin strongly inhibited the expression of gp91(phox)mRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-alpha production, diapocynin had a much more pronounced effect, on both TNF-alpha and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91(phox) mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched. (C) 2010 Elsevier Inc. All rights reserved.

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Myeloperoxidase inhibitory and radical scavenging activities of flavones from Pterogyne nitens

Two new flavone glucosides, nitensosides A and B (1, 2), together with four known compounds, sorbifolin (3), sorbifolin 6-O-β-glucopyranoside (4), pedalitin (5), and pedalitin 6-O-β-glucopyranoside (6) were isolated from Pterogyne nitens. Their structures were elucidated from 1D and 2D NMR analysis, as well as by high resolution mass spectrometry. All the isolated flavones were evaluated for their myeloperoxidase (MPO) inhibitory activity. The most active compound, pedalitin, exhibited IC 50 value of 3.75 nM on MPO. Additionally, the radical-scavenging capacity of flavones 1-6 was evaluated towards ABTS and DPPH radicals and compared to standard compounds quercetin and Trolox®. © 2008 Pharmaceutical Society of Japan.

The importance of myeloperoxidase in apocynin-mediated NADPH oxidase inhibition

Apocynin is widely used as an inhibitor of the NADPH oxidase. Since myeloperoxidase (MPO) has been considered as essential for the mechanism of action of apocynin, here we used cells with different levels of MPO and compared their sensitivity to apocynin. HL-60 cells were differentiated with DMSO or IFN γ /TNF α and compared with peripheral mononuclear (PBMC) and polymorphonuclear cells (PMN). The relative MPO activity was PBMC = HL60 DMSO < HL60 IFN γ < PMN. Apocynin inhibited the intracellular reactive oxygen species production by PMN (80%) and IFN γ /TNF α -differentiated HL-60 cells (45%) but showed a minor effect in PBMC and DMSO differentiated HL-60 cells (20%). The addition of azide decreased the efficiency of apocynin in PMN and the addition of peroxidase increased the inhibition in PBMC. We also determined the gene expression of the components gp91phox, p47phox, p22phox and p67phox in the resting cells. Apocynin did not change gp91phox, p47phox or p22phox gene expression in nonstimulated PBMC, HL60 DMSO, HL60 IFN γ /TNF α , and PMN and has a subtle increase in p67phox in HL60 IFN γ /TNF α . The results from this work suggest that a rational search for better inhibitors of NADPH oxidase in leukocytes should include a correlation with their affinity as substrates for MPO.

OXIDATIVE STRESS BASED STRATEGIES FOR ENHANCING THE EFFICACY OF HISTONE DEACETYLASE INHIBITORS (HDACi)

Histone deacetylase inhibitors (HDACi) are anti-cancer drugs that primarily act upon acetylation of histones, however they also increase levels of intracellular reactive oxygen species (ROS). We hypothesized that agents that cause oxidative stress might enhance the efficacy of HDACi. To test this hypothesis, we treated acute lymphocytic leukemia cells (ALL) with HDACi and adaphostin (ROS generating agent). The combination of two different HDACi (vorinostat or entinostat) with adaphostin synergistically induced apoptosis in ALL. This synergistic effect was blocked when cells were pre-treated with the caspase-9 inhibitor, LEHD. In addition, we showed that loss of the mitochondrial membrane potential is the earliest event observed starting at 12 h. Following this event, we observed increased levels of superoxide at 16 h, and ultimately caspase-3 activation. Pre-treatment with the antioxidant N-acetylcysteine (NAC) blocked ROS generation and reversed the loss of mitochondrial membrane potential for both combinations. Interestingly, DNA fragmentation and caspase-3 activity was only blocked by NAC in cells treated with vorinostat-adaphostin; but not with entinostat-adaphostin. These results suggest that different redox mechanisms are involved in the induction of ROS-mediated apoptosis. To further understand these events, we studied the role of the antioxidants glutathione (GSH) and thioredoxin (Trx). We found that the combination of entinostat-adaphostin induced acetylation of the antioxidant thioredoxin (Trx) and decreased intracellular levels of GSH. However, no effect on Trx activity was observed in either combination. In addition, pre-treatment with GSH ethyl ester, a soluble form of GSH, did not block DNA fragmentation. Together these results suggested that GSH and Trx are not major players in the induction of oxidative stress. Array data examining the expression of genes involved in oxidative stress demonstrated a differential regulation between cells treated with vorinostat-adaphostin and entinostat-adaphostin. Some of the genes differentially expressed between the combinations include aldehyde oxidase 1, glutathione peroxidase-5, -6, peroxiredoxin 6 and myeloperoxidase. Taken together, these experimental results indicate that the synergistic activity of two different HDACi with adaphostin is mediated by distinct redox mechanisms in ALL cells. Understanding the mechanism involved in these combinations will advance scientific knowledge of how the action of HDACi could be augmented in leukemia models. Moreover, this information could be used for the development of effective clinical trials combining HDACi with other anticancer agents.

The PG500 series : novel heparan sulfate mimetics as potent angiogenesis and heparanase inhibitors for cancer therapy

Heparan sulfate mimetics, which we have called the PG500 series, have been developed to target the inhibition of both angiogenesis and heparanase activity. This series extends the technology underpinning PI-88, a mixture of highly sulfated oligosaccharides which reached Phase III clinical development for hepatocellular carcinoma. Advances in the chemistry of the PG500 series provide numerous advantages over PI-88. These new compounds are fully sulfated, single entity oligosaccharides attached to a lipophilic moiety, which have been optimized for drug development. The rational design of these compounds has led to vast improvements in potency compared to PI-88, based on in vitro angiogenesis assays and in vivo tumor models. Based on these and other data, PG545 has been selected as the lead clinical candidate for oncology and is currently undergoing formal preclinical development as a novel treatment for advanced cancer.

Inhibitors to optimal project portfolio selection

The selection of projects and programs of work is a key function of both public and private sector organisations. Ideally, projects and programs that are selected to be undertaken are consistent with strategic objectives for the organisation; will provide value for money and return on investment; will be adequately resourced and prioritised; will not compete with general operations for resources and not restrict the ability of operations to provide income to the organisation; will match the capacity and capability of the organisation to deliver; and will produce outputs that are willingly accepted by end users and customers. Unfortunately,this is not always the case. Possible inhibitors to optimal project portfolio selection include: processes that are inconsistent with the needs of the organisation; reluctance to use an approach that may not produce predetermined preferences; loss of control and perceived decision making power; reliance on quantitative methods rather than qualitative methods for justification; ineffective project and program sponsorship; unclear project governance, processes and linkage to business strategies; ignorance, taboos and perceived effectiveness; inadequate education and training about the processes and their importance.