Generation of anti-complement "prodrugs": cleavable reagents for specific delivery of complement regulators to disease sites

Expression of biologically active molecules as fusion proteins with antibody Fc can substantially extend the plasma half-life of the active agent but may also influence function. We have previously generated a number of fusion proteins comprising a complement regulator coupled to Fc and shown that the hybrid molecule has a long plasma half-life and retains biological activity. However, several of the fusion proteins generated had substantially reduced biological activity when compared with the native regulator or regulator released from the Fc following papain cleavage. We have taken advantage of this finding to engineer a prodrug with low complement regulatory activity that is cleaved at sites of inflammation to release active regulator. Two model prodrugs, comprising, respectively, the four short consensus repeats of human decay accelerating factor (CD55) linked to IgG4 Fc and the three NH2-terminal short consensus repeats of human decay accelerating factor linked to IgG2 Fc have been developed. In each, specific cleavage sites for matrix metalloproteinases and/or aggrecanases have been incorporated between the complement regulator and the Fc. These prodrugs have markedly decreased complement inhibitory activity when compared with the parent regulator in vitro. Exposure of the prodrugs to the relevant enzymes, either purified, or in supernatants of cytokine-stimulated chondrocytes or in synovial fluid, efficiently cleaved the prodrug, releasing active regulator. Such agents, having negligible systemic effects but active at sites of inflammation, represent a paradigm for the next generation of anti-C therapeutics.

Mapping CD55 function: the structure of two pathogen-binding domains at 1.7 A

Decay-accelerating factor (CD55), a regulator of the alternative and classical pathways of complement activation, is expressed on all serum-exposed cells. It is used by pathogens, including many enteroviruses and uropathogenic Escherichia coli, as a receptor prior to infection. We describe the x-ray structure of a pathogen-binding fragment of human CD55 at 1.7 A resolution containing two of the three domains required for regulation of human complement. We have used mutagenesis to map biological functions onto the molecule; decay-accelerating activity maps to a single face of the molecule, whereas bacterial and viral pathogens recognize a variety of different sites on CD55.

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.

Mapping antimalarial pharmacophores as a useful tool for the rapid discovery of drugs effective in vivo: design, construction, characterization, and pharmacology of metaquine

Resistant strains of Plasmodium falciparum and the unavailability of useful antimalarial vaccines reinforce the need to develop new efficacious antimalarials. This study details a pharmacophore model that has been used to identify a potent, soluble, orally bioavailable antimalarial bisquinoline, metaquine (N,N'-bis(7-chloroquinolin-4-yl)benzene-1,3-diamine) (dihydrochloride), which is active against Plasmodium berghei in vivo (oral ID50 of 25 mu mol/kg) and multidrug-resistant Plasmodium falciparum K1 in vitro (0.17 mu M). Metaquine shows strong affinity for the putative antimalarial receptor, heme at pH 7.4 in aqueous DMSO. Both crystallographic analyses and quantum mechanical calculations (HF/6-31+G*) reveal important regions of protonation and bonding thought to persist at parasitic vacuolar pH concordant with our receptor model. Formation of drug-heme adduct in solution was confirmed using high-resolution positive ion electrospray mass spectrometry. Metaquine showed strong binding with the receptor in a 1: 1 ratio (log K = 5.7 +/- 0.1) that was predicted by molecular mechanics calculations. This study illustrates a rational multidisciplinary approach for the development of new 4-aminoquinoline antimalarials, with efficacy superior to chloroquine, based on the use of a pharmacophore model.

Nitrogen management is essential to prevent tropical oil palm plantations from causing ground-level ozone pollution

More than half the world's rainforest has been lost to agriculture since the Industrial Revolution. Among the most widespread tropical crops is oil palm (Elaeis guineensis): global production now exceeds 35 million tonnes per year. In Malaysia, for example, 13% of land area is now oil palm plantation, compared with 1% in 1974. There are enormous pressures to increase palm oil production for food, domestic products, and, especially, biofuels. Greater use of palm oil for biofuel production is predicated on the assumption that palm oil is an "environmentally friendly'' fuel feedstock. Here we show, using measurements and models, that oil palm plantations in Malaysia directly emit more oxides of nitrogen and volatile organic compounds than rainforest. These compounds lead to the production of ground-level ozone (O-3), an air pollutant that damages human health, plants, and materials, reduces crop productivity, and has effects on the Earth's climate. Our measurements show that, at present, O-3 concentrations do not differ significantly over rainforest and adjacent oil palm plantation landscapes. However, our model calculations predict that if concentrations of oxides of nitrogen in Borneo are allowed to reach those currently seen over rural North America and Europe, ground-level O-3 concentrations will reach 100 parts per billion (10(9)) volume (ppbv) and exceed levels known to be harmful to human health. Our study provides an early warning of the urgent need to develop policies that manage nitrogen emissions if the detrimental effects of palm oil production on air quality and climate are to be avoided.

Direct estimates of emissions from the megacity of Lagos

We report here top-down emissions estimates for an African megacity. A boundary layer circumnavigation of Lagos, Nigeria was completed using the FAAM BAe146 aircraft as part of the AMMA project. These observations together with an inferred boundary layer height allow the flux of pollutants to be calculated. Extrapolation gives annual emissions for CO, NOx, and VOCs of 1.44 Tg yr(-1), 0.03 Tg yr(-1) and 0.37 Tg yr(-1) respectively with uncertainties of (+250)/(-60%). These inferred emissions are consistent with bottom-up estimates for other developing megacities and are attributed to the evaporation of fuels, mobile combustion and natural gas emissions.

Comparative characterisation by atomic force microscopy and ellipsometry of soft and solid thin films

Ellipsometry and atomic force microscopy (AFM) were used to study the film thickness and the surface roughness of both 'soft' and solid thin films. 'Soft' polymer thin films of polystyrene and poly(styrene-ethylene/butylene-styrene) block copolymer were prepared by spin-coating onto planar silicon wafers. Ellipsometric parameters were fitted by the Cauchy approach using a two-layer model with planar boundaries between the layers. The smooth surfaces of the prepared polymer films were confirmed by AFM. There is good agreement between AFM and ellipsometry in the 80-130 nm thickness range. Semiconductor surfaces (Si) obtained by anisotropic chemical etching were investigated as an example of a randomly rough surface. To define roughness parameters by ellipsometry, the top rough layers were treated as thin films according to the Bruggeman effective medium approximation (BEMA). Surface roughness values measured by AFM and ellipsometry show the same tendency of increasing roughness with increased etching time, although AFM results depend on the used window size. The combined use of both methods appears to offer the most comprehensive route to quantitative surface roughness characterisation of solid films. Copyright (c) 2007 John Wiley & Sons, Ltd.

Ellipsometric study of adsorption on nanopatterned block copolymer substrates

We report ellipsometrically obtained adsorption isotherms for a carefully chosen test liquid on block copolymer films of Kraton G1650, compared with adsorption isotherms on homogeneous films of the constituent polymers. Standard atomic force microscopy images imply the outer surface of Kraton G1650 is chemically patterned on the nanoscale, but this could instead be a reflection of structure buried beneath a 10 nm layer of the lower energy component. Our test liquid was chosen on the basis that it did not dissolve in either component and in addition that it was nonwetting on the lower energy polymer while forming thick adsorbed films on pure substrates of the higher energy component. Our ellipsometry data for Kraton G1650 rule out the presence of segregation by the lower energy constituent to the outer surface, implying a mixed surface consistent with Cassie's law. We discuss implications of our findings and related work for the outer surface structures of block copolymer films.

Carbohydrate-based therapeutics

In recent years there has been a resurgence of interest in the biological roles of carbohydrates and as a result it is now known that carbohydrates are involved in a vast array of disease processes. This review summarises progress in the development of carbohydrate-based therapeutics that involve: inhibition of carbohydrate-lectin interactions; immunisation, using monoclonal antibodies for carbohydrate antigens; inhibition of enzymes that synthesise disease-associated carbohydrates; replacement of carbohydrate-processing enzymes; targeting of drugs to specific disease cells via carbohydrate-lectin interactions; carbohydrate based anti-thrombotic agents.

Flavonoids: antioxidants or signalling molecules?

Many studies are accumulating that report the neuroprotective, cardioprotective, and chemopreventive actions of dietary flavonoids. While there has been a major focus on the antioxidant properties, there is an emerging view that flavonoids, and their in vivo metabolites, do not act as conventional hydrogen-donating antioxidants but may exert modulatory actions in cells through actions at protein kinase and lipid kinase signalling pathways. Flavonoids, and more recently their metabolites, have been reported to act at phosphoinositide 3-kinase (PI 3-kinase), Akt/protein kinase B (Akt/PKB), tyrosine kinases, protein kinase C (PKC), and mitogen activated protein kinase (MAP kinase) signalling cascades. Inhibitory or stimulatory actions at these pathways are likely to affect cellular function profoundly by altering the phosphorylation state of target molecules and by modulating gene expression. A clear understanding of the mechanisms of action of flavonoids, either as antioxidants or modulators of cell signalling, and the influence of their metabolism on these properties are key to the evaluation of these potent biomolecules as anticancer agents, cardioprotectants, and inhibitors of neurodegeneration (C) 2004 Elsevier Inc. All rights reserved.

Zinc-histidine complex protects cultured cortical neurons against oxidative stress-induced damage

The levels of zinc in the brain are directly affected by dietary zinc and deficiency has been associated with alcohol withdrawal seizures, excitotoxicity, impaired learning and memory and an accelerated rate of dysfunction in aged brain. Although zinc is essential for a healthy nervous system, high concentrations of zinc are neurotoxic, thus it is important to identify the most effective forms of zinc for treatment of conditions of the central nervous system. Accumulating evidence suggests that zinc-histidine complex (Zn(HiS)(2)) has greater biological potency and enhanced bioavailability compared with other zinc salts and also has antioxidant potential. Therefore, in this study we investigated the ability of zinc-histidine to protect cultured cortical neurons against hydrogen peroxide-induced damage. Pre-treating neurons for 18h with subtoxic concentrations of zinc-histidine (5-25 muM) improved neuronal viability and strongly inhibited hydrogen peroxide-induced (75 muM, 30 min) cell damage as assessed by MTT turnover and morphological analysis 24 It later. Low concentrations of zinc-histidine were more neuroprotective than zinc chloride. There was evidence of an anti-apoptotic mechanism of action as zinc-histidine inhibited hydrogen peroxide-induced caspase-3 activation and c-jun-N-terminal kinase phosphorylation. In summary, zinc supplementation with zinc-histidine protects cultured neurons against oxidative insults and inhibits apoptosis which suggests that zinc-histidine may be beneficial in the treatment of diseases of the CNS associated with zinc deficiency. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Activation of pro-survival Akt and ERK1/2 signalling pathways underlie the anti-apoptotic effects of flavanones in cortical neurons

There is growing interest in the potential beneficial effects of flavonoids in the aging and diseased brain. We have investigated the potential of the flavanone hesperetin and two of its metabolites, hesperetin-7-O-beta-D-glucuronide and 5-nitro-hesperetin, to inhibit oxidative stress-induced neuronal apoptosis. Exposure of cortical neurons to hydrogen peroxide led to the activation of apoptosis signal-regulating kinase 1 via its de-phosphorylation at Ser963, the phosphorylation of c-jun N-terminal kinase and c-Jun (Ser73) and the activation of caspase 3 and caspase 9. Whilst hesperetin glucuronide failed to exert protection, both hesperetin and 5-nitro-hesperetin were effective at preventing neuronal apoptosis via a mechanism involving the activation/phosphorylation of both Akt/protein kinase B and extracellular signal-regulated kinase 1 and 2 (ERK1/2). Protection against oxidative injury and the activation of Akt and ERK1/2 followed a bell-shaped response and was most apparent at 100 nmol/L concentrations. The activation of ERK1/2 and Akt by flavanones led to the inhibition of the pro-apoptotic proteins, apoptosis signal-regulating kinase 1, by phosphorylation at Ser83 and Bad, by phosphorylation at both Ser136 and Ser112 and to the inhibition of peroxide-induced caspase 9 and caspase 3 activation. Thus, flavanones may protect neurons against oxidative insults via the modulation of neuronal apoptotic machinery.

Inhibition of cellular proliferation by the genistein metabolite 5,7,3',4'-tetrahydroxyisoflavone is mediated by DNA damage and activation of the ATR signalling pathway

The cellular actions of genistein, and its in vivo metabolites, are believed to mediate the decreased risk of breast cancer associated with high soy consumption. The genistein metabolite, 5,7,3',4'-tetrahydroxyisoflavone (THIF), induced G2-M cell cycle arrest in T47D tumorigenic breast epithelial cells via a mechanism involving the activation of ataxia telangiectasia and Rad3-related kinase (ATR) via its phosphorylation at Ser(428). This activation of ATR appeared to result from THIF-induced increases in intracellular oxidative stress, a depletion of cellular GSH and an increase in DNA strand breakage. THIF treatment also led to an inhibition of cdc2, which was accompanied by the phosphorylation of both p53 (Ser(15)) and Chk1 (Ser(296)) and the de-activation of cdc25C phosphatase. We suggest the anti-proliferative actions of THIF may be mediated by initial oxidative DNA damage, activation of ATR and downstream regulation of the p53 and Chk1 pathways leading to cell cycle arrest in G2-M. This may represent one mechanism by which genistein exerts its cellular activity in vivo. (c) 2007 Elsevier Inc. All rights reserved.