Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

NS1 proteins of avian influenza A viruses can act as antagonists of the human alpha/beta interferon response

Many viruses, including human influenza A virus, have developed strategies for counteracting the host type I interferon (IFN) response. We have explored whether avian influenza viruses were less capable of combating the type I IFN response in mammalian cells, as this might be a determinant of host range restriction. A panel of avian influenza viruses isolated between 1927 and 1997 was assembled. The selected viruses showed variation in their ability to activate the expression of a reporter gene under the control of the IFN-beta promoter and in the levels of IFN induced in mammalian cells. Surprisingly, the avian NS1 proteins expressed alone or in the genetic background of a human influenza virus controlled IFN-beta induction in a manner similar to the NS1 protein of human strains. There was no direct correlation between the IFN-beta induction and replication of avian influenza viruses in human A549 cells. Nevertheless, human cells deficient in the type I IFN system showed enhanced replication of the avian viruses studied, implying that the human type I IFN response limits avian influenza viruses and can contribute to host range restriction.

AT(1) receptor ligands: virtual-screening-based design with TOPP descriptors, synthesis, and biological evaluation of pyrrolidine derivatives

As a continuing effort to establish the structure-activity relationships (SARs) within the series of the angiotensin II antagonists (sartans), a pharmacophoric model was built by using novel TOPP 3D descriptors. Statistical values were satisfactory (PC4: r(2)=0.96, q(2) ((5) (random) (groups))=0.84; SDEP=0.26) and encouraged the synthesis and consequent biological evaluation of a series of new pyrrolidine derivatives. SAR together with a combined 3D quantitative SAR and high-throughput virtual screening showed that the newly synthesized 1-acyl-N-(biphenyl-4-ylmethyl)pyrrolidine-2-carboxamides may represent an interesting starting point for the design of new antihypertensive agents. In particular, biological tests performed on CHO-hAT(1) cells stably expressing the human AT(1) receptor showed that the length of the acyl chain is crucial for the receptor interaction and that the valeric chain is the optimal one.

The effect of carrier substrate, dose rate and time of application on biocontrol efficacy of fungal antagonists against Armillaria root rot of strawberry plants.

In a glasshouse experiment using potted strawberry plants (cv. Cambridge Favourite) as hosts, the effect of selected fungal antagonists grown on 25 or 50 g of mushroom compost containing autoclaved mycelia of Agaricus bisporus, or wheat bran was evaluated against Armillaria mellea. Another glasshouse experiment tested the effect of application time of the antagonists in relation to inoculations with the pathogen. A significant interaction was found between the antagonists, substrates and dose rates. All the plants treated with Chaetomium olivaceum isolate Co on 50 g wheat bran survived until the end of the experiment which lasted 482 days, while none of them survived when this antagonist was added to the roots of the plants on 25 g wheat bran or 25 or 50 g mushroom compost. Dactylium dendroides isolate SP had a similar effect, although with a lower host survival rate of 33.3%. Trichoderma hamatum isolate Tham 1 and T. harzianum isolate Th23 protected 33.3% of the plants when added on 50 g and none when added on 25 g of either substrate, while 66.7% of the plants treated with T. harzianum isolate Th2 on 25 g, or T viride isolate TO on 50 g wheat bran, survived. Application of the antagonists on mushroom compost initially resulted in development of more leaves and healthier plants, but this effect was not sustained. Eventually, plants treated with the antagonists on wheat bran had significantly more leaves and higher health scores. The plants treated with isolate Th2 and inoculated with Armillaria at the same time had a survival rate of 66.7% for the duration of the experiment (475 days), while none of them survived that long when the antagonist and pathogen were applied with an interval of 85 days in either sequence. C. olivaceum isolate Co showed a protective effect only, as 66.7% of the plants survived when they were treated with the antagonist 85 days before inoculation with the pathogen, while none of them survived when the antagonist and pathogen were applied together or the infection preceded protection.

Cultural techniques for improvement in biocontrol potential of fungal antagonists for biological control of Armillaria root rot of strawberry plants under glasshouse conditions

Several in vitro and in vivo experiments were conducted to develop an effective technique for culturing potential fungal antagonists (isolates of Trichoderma harzianum, Dactylium dendroides, Chaetomium olivaceum and one unidentified fungus) selected for activity against Armillaria mellea. The antagonists were inoculated onto (1) live spawn of the oyster mu shroom (Pleurotus ostreatus), (2) extra-moistened or sucrose-enriched mushroom composts containing living or autoclaved mycelia of P. ostreatus or Agaricus bisporus (button mushroom), (3) pasteurized compost with or without A. bisporus mycelium, wheat bran, wheat germ and (4) spent mushroom composts with living mycelia of A. bisporus, P. ostreatus or Lentinus edodes (the Shiitake mushroom). In one experiment, a representative antagonist (isolate Th2 of T. harzianum) was grown together with the A. bisporus mycelium, while in another one, the antagonist was first grown on wheat germ or wheat bran and then on mushroom compost with living mycelium of A. bisporus. Some of the carrier substrates were then added to the roots of potted strawberry plants in the glasshouse to evaluate their effectiveness against the disease. The antagonists failed to grow on the spawn of P. ostreatus even after reinoculations and prolonged incubation. Providing extra moisture or sucrose enrichment also did not improve the growth of Th2 on mushroom composts in the presence of living mycelia of A. bisporus or P. ostreatus. The antagonist, however, grew rapidly and extensively on mushroom compost with autoclaved mycelia, and also on wheat germ and wheat bran. Colonization of the substrates by the antagonist was positively correlated with its effectiveness in the glasshouse studies. Whereas only 33.3% of the inoculated control plants survived in one experiment monitored for 560 days, 100% survival was achieved when Th2 was applied on wheat germ or wheat bran. Growth of the antagonist alone on pasteurized or sterilized compost (without A. bisporus mycelia) and simultaneous growth of the antagonist and mushroom on pasteurized compost did not improve survival over the inoculated controls, but growth over mushroom compost with the living mycelium resulted in 50% survival rate. C. olivaceum isolate Co was the most effective, resulting in overall survival rate of 83.3% compared with only 8.3% for the inoculated and 100% for the uninoculated (healthy) controls. This antagonist gave the highest survival rate of 100% on spent mushroom compost with L. edodes. T harzianum isolate Th23, with 75% survival rate, was the most effective on spent mushroom compost with P. ostreatus, while D. dendroides isolate SP resulted in equal survival rates of 50% on all the three mushroom composts.

Comparisons between the in vitro and in vivo efficacies of potential fungal antagonists of Armillaria mellea

Seventeen fungal isolates were tested in vitro as potential antagonists of two isolates of the root rot pathogen, Armillaria mellea. Some of the isolates were also added on mushroom composts with living mycelia to the roots of Armillaria-inoculated potted strawberry plants in the glasshouse to find out if they had the same degree of efficacy against the disease. Dactylium dendroides isolate SP was the most effective in reducing mycelial growth of A. mellea isolate 1 (Am1), followed by Trichoderma harzianum isolate Th2 and T. viride isolate Tv4. Th2, Th22, Tv3 and SP grew extensively over Am1 colonies, disintegrating the rhizomorphs. Isolate Tham1 of T hamatum was the most effective in reducing mycelial growth of A. mellea isolate 2 (Am2), followed by Tv3. Th12, Th22, Tv1, Tv3 and SP inhibited the initiation and growth of rhizomorphs of Am2. Regeneration tests showed that both Am1 and Am2 attacked by Trichoderma isolates and SP were no longer viable. Th23 and SP were almost as effective in vivo as in vitro. But isolate Co of Chaetomium olivaceum, which was ineffective in vitro, was found effective in vivo. Conversely, Th2, which exhibited good antagonistic activity in vitro, performed poorly in vivo. These results show that the in vitro and in vivo efficacies of potential antagonists may not necessarily be closely correlated. Hence, there is a danger that potentially effective isolates may be discarded if decisions are made only on the basis of preliminary screening tests carried out under laboratory conditions.

Allosteric effects of antagonists on signalling by the chemokine receptor CCR5

Antagonists of the chemokine receptor, CCRS, may provide important new drugs for the treatment of HIV-1. In this study we have examined the mechanism of action of two functional antagonists of the chemokine receptor CCRS (UK-396,794, UK-438,235) in signalling and internalisation assays using CHO cells expressing CCR5. Both compounds were potent inverse agonists versus agonist-independent [S-3]GTP gamma S binding to membranes of CHO cells expressing CCR5. Both compounds also acted as allosteric inhibitors of CCL5 (RANTES) and CCL8 (MCP-2) -stimulated [S-35]GTP gamma S binding to CHO-CCR5 membranes, reducing the potency and maximal effects of the two chemokines. The data are consistent with effects of the allosteric inhibitors on both the binding and signalling of the chemokines. Both compounds inhibited CCR5 internalisation triggered by chemokines. When CHO-CCR5 cells were treated with either of the two compounds for prolonged periods of time (24 h) an increase (similar to 15%) in cell surface CCRS was detected. (C) 2007 Elsevier Inc. All rights reserved

Endocannabinoid receptor antagonists: potential for obesity treatment

Obesity has been described as a global epidemic. Its increasing prevalence is matched by growing costs, not only to the health of the individual, but also to the medical services required to treat a range of obesity-related diseases. In most instances, obesity is a product of progressively less energetic lifestyles and the over-consumption of readily available, palatable, and highly caloric foods. Past decades have seen massive investment in the search for effective anti-obesity therapies, so far with limited success. An important part of the process of developing new pharmacologic treatments for obesity lies in improving our understanding of the psychologic and physiologic processes that govern appetite and bodyweight regulation. Recent discoveries concerning the endogenous cannabinoids are beginning to give greater insight into these processes. Current research indicates that endocannabinoids may be key to the appetitive and consummatory aspects of eating motivation, possibly mediating the craving for and enjoyment of the most desired, most fattening foods. Additionally, endocannabinoids appear to modulate central and peripheral processes associated with fat and glucose metabolism. Selective cannabinoid receptor antagonists have been shown to suppress the motivation to eat, and preferentially reduce the consumption of palatable, energy-dense foods. Additionally, these agents act to reduce adiposity through metabolic mechanisms that are independent of changes in food intake. Given the current state of evidence, we conclude that the endocannabinoids represent an exciting target for new anti-obesity therapies.

Production of angiotensin-I-converting enzyme (ACE) inhibitory activity in milk fermented with probiotic strains: Effects of calcium, pH and peptides on the ACE-inhibitory activity

Recently, probiotic fermented milk products have raised interest regarding their potential anti-hypertensive activity mainly due to the production of angiotensin-I-converting enzyme (ACE) inhibitory peptides. Ionic calcium released upon milk acidification during fermentation is also known to exert hypotensive activity. Thus, the main aim of this study was to screen probiotic strains for their ability to induce ACE-inhibitory activity upon fermentation of milk. The relationship of ACE-inhibitory activity percentage (ACEi%) with cell growth, pH, degree of hydrolysis and the concentration of ionic calcium released during the fermentation was also investigated. Compared with other lactic acid bacteria, Lactobacillus casei YIT 9029 and Bifidobacterium bifidum MF 20/5 were able to induce strong ACE-inhibitory activity. Furthermore, it was found that the ionic calcium released during milk fermentation could contribute to the ACE-inhibitory activity. These findings will contribute to the development of new probiotic dairy products with anti-hypertensive activity.

Production of angiotensin converting enzyme inhibitory peptides from β-lactoglobulin and casein derived peptides: an integrative approach

Angiotensin I-converting enzyme (ACE) inhibition is one of the mechanisms by which reduction in blood pressure is exerted. Whey proteins are a rich source of ACE inhibitory peptides and have shown a blood pressure reduction effect i.e. antihypertensive activity. The aim of this work was to develop a simplified process using a combination of adsorption and microfiltration steps for the production of hydrolysates from whey with high ACE inhibitory activity and potency; the latter was measured as the IC50, which is the peptide concentration required to reduce ACE activity by half. This process integrates the selective separation of β-lactoglobulin and casein derived peptides (CDP) from rennet whey and their hydrolysis, which results in partially pure, less complex hydrolysates with high bioactive potency. Hydrolysis was carried out with protease N ‘Amano’ in a thermostatically controlled membrane reactor operated in a batch mode. By applying the integrative approach it was possible to produce from the same feedstock two different hydrolysates that exhibited high ACE inhibition. One hydrolysate was mainly composed of casein-derived peptides with IC50= 285 μg/mL. In this hydrolysate we identified the well known potent ACE-I and anti-hypertensive tri-peptide Ile-Pro-Pro (IPP) and another novel octa-peptide Gln-Asp-Lys-Thr-Glu-Ile-Pro-Thr (QDKTEIPT). The second hydrolysate was mainly composed of β-lactoglobulin derived peptides with IC50=128 µg/mL. This hydrolysate contained a tetra-peptide (Ile-Ile-Ala-Glu) IIAE as one of the two major peptides. A further advantage to this process is that enzyme activity was substantially increased as enzyme product inhibition was reduced.

'A theatre of ruins'. Edward Bond & Samuel Beckett: theatrical antagonists

The playwright Edward Bond has long made known his antagonism to dramatists allied to Martin Esslin’s Theatre of the Absurd. The work of Samuel Beckett has come in for particular criticism by Bond. Using published writings (and unpublished correspondence between myself and Bond), I hope to trace the development of this antagonism between ‘Bondian’ and ‘Beckettian’ views of theatre. However, this article will also set out to argue that both early work such as The Pope’s Wedding (1962), and more recent work such as Coffee (1995), make use of motifs, characters and ideas from Beckett’s theatre. The article will set out provisional reasons why Bond, despite his misgivings, is not averse to incorporating elements from Beckett’s ‘theatre of ruins’, as he terms it, into his own work.

Discovery of novel GPVI receptor antagonists by structure-based repurposing.

Inappropriate platelet aggregation creates a cardiovascular risk that is largely managed with thienopyridines and aspirin. Although effective, these drugs carry risks of increased bleeding and drug 'resistance', underpinning a drive for new antiplatelet agents. To discover such drugs, one strategy is to identify a suitable druggable target and then find small molecules that modulate it. A good and unexploited target is the platelet collagen receptor, GPVI, which promotes thrombus formation. To identify inhibitors of GPVI that are safe and bioavailable, we docked a FDA-approved drug library into the GPVI collagen-binding site in silico. We now report that losartan and cinanserin inhibit GPVI-mediated platelet activation in a selective, competitive and dose-dependent manner. This mechanism of action likely underpins the cardioprotective effects of losartan that could not be ascribed to its antihypertensive effects. We have, therefore, identified small molecule inhibitors of GPVI-mediated platelet activation, and also demonstrated the utility of structure-based repurposing.