Drug Discovery Opinion

Although carbohydrates were previously thought of as little more than an energy source, it is now recognised that they play a key role in many biochemical processes including intercellular recognition, immune function, fertilisation and certain types of cancer. From a synthetic viewpoint, the structural complexity that makes carbohydrates important in so many biological processes poses significant challenges. Unlike oligonucleotides and peptides, carbohydrates can form branched as well as linear structures, and there are a staggering number of ways in which monosaccharides can be combined. Increasingly powerful and versatile methods have been developed to allow the synthesis of pure oligosaccharides in the laboratory, but the process requires regioselective protection of hydroxyl groups as well as stereoselective assembly of glycosidic linkages and is technically difficult and very time consuming. Speaking at the annual meeting of the American Chemical Society in Salt Lake City, Dr Peter H Seeberger has described the development of a fully automated carbohydrate synthesiser that should make complex carbohydrates more accessible. A simple cleavage, deprotection and purification protocol provides rapid access to naturally occurring and synthetic oligosaccharides and is suitable for use by non-experts.

One application that Seeberger’s group have been addressing with the new technology is the development of a vaccine against malaria. Fatalities caused by the malaria parasite, Plasmodium falciparum, are thought to result, at least in part, from a reaction to the malaria toxin, glycosylphosphatidylinositol (GPI). Anti-GPI vaccination was found to protect against fatality in mice and clinical trials of an anti-toxin vaccine to protect against the inflammation and anaemia associated with malarial infection are scheduled for 2010 in Mozambique and Tanzania. Seeberger also believes that carbohydrate-based vaccines could be used against other serious infectious diseases, including antibiotic-resistant infections and HIV.

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Cold sores are fluid-filled blisters caused mainly by the highly contagious herpes simplex virus type 1 (HSV-1), which is passed on by close skin contact with someone with a cold sore. Most children come into contact with the virus by the age of five but many show no symptoms until puberty. HSV-1 is not cleared from the body by the immune system and, following initial infection of cells of the epidermis, the virus travels to the nerve root ganglia where it becomes dormant and causes no symptoms. As a result of the primary infection, the body produces antibodies against the virus – the majority of adults have antibodies against HSV-1 and 20 per cent of these individuals will have recurrent attacks of cold sores throughout their lives. The causes of reactivation are uncertain but are thought to include physical or psychological stress as well as changes to immune function.

Writing in the journal Nature Immunology, scientists from Canada and the US have now described a novel cellular process that helps the body to fight HSV-1 infection. In mouse cells infected with HSV-1, the researchers discovered a previously unknown type of autophagosome originating from the nuclear membrane. When conditions of low-grade fever were replicated by hyperthermia, or the cells were exposed to the pyrogenic cytokine, interleukin 1β, autophagy and vacuolar processing of viral peptides was observed. Viral peptides in autophagosomes were further processed by the proteasome, suggesting a complex interaction between the vacuolar and previously described MHC class I presentation pathways.

The research team hope that their discovery might eventually lead to novel therapies for HSV-1 infections, as well as for infections caused by other pathogens that are able to evade the immune system.

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Liquorice, which is used as a traditional medicine, a culinary spice and an ingredient of confectionary, is an expectorant and mild laxative, has antiviral properties and also increases blood pressure at high doses. One of the active ingredients of liquorice-root extract is the natural sweetener glycyrrhizin, which is over 50 times sweeter than sucrose. Two recent reports describe additional properties of liquorice.

Writing in the Journal of Clinical Investigation, researchers at Vanderbilt University Medical Center suggest that glycyrrhizic acid may offer a new approach to preventing colorectal cancer (CRC), the second leading cause of cancer deaths in the United States. Prostaglandin E₂ (PGE₂) has been shown to promote CRC progression and both non-selective cyclooxygenase (COX) inhibitors and selective COX-2 inhibitors, which block production of PGE₂, reduce the number and size of colonic adenomas. Although COX-2 inhibition, which reduces the number and size of colon polyps in both mice and patients with a predisposition to colon cancer, is a promising target for chemoprevention of CRC, both non-selective NSAIDs and selective COX-2 inhibitors have side effects that limit their prophylactic use. The Vanderbilt team had previously shown that inhibiting 11β–hydroxysteroid dehydrogenase type II (11βHSD2) in the kidney suppresses COX-2 expression and, since the colon is one of the only other organs with high expression of 11βHSD2, speculated that this enzyme might play a role in colorectal cancer progression. Expression of 11βHSD2, which converts active glucocorticoids to inactive keto-forms, was found to be increased in polyps found in both mice and humans and correlated with COX-2 expression and activity. Inhibition of 11βHSD2 with glycyrrhizic acid was found to inhibit COX-2–mediated PGE₂ production in tumours, and to prevent polyp formation, tumour growth, and metastasis in mice.

Inhibition of 11βHSD2 also increases cortisol levels in the kidney and contributes to the increases in blood pressure caused by large doses of glycyrrhizic acid. The team did not see any increases in blood pressure in the treated mice, but believe that this relatively benign side effect could easily be treated by diuretics. The team now want to design more potent and selective inhibitors of 11βHSD2 and explore the role of 11βHSD2 in other cancers.

Chemists in Taiwan have also recently warned that both glycyrrhizin and liquorice extracts can block the absorption of cyclosporine, a drug used to prevent rejection of organ transplants and as a treatment for rheumatoid arthritis. Speaking at the annual meeting of the American Chemical Society in Salt Lake City, Dr Pei-Dawn Lee Chao described how levels of cyclosporine were reduced in laboratory rats when co-administered with liquorice. Although it is not known how much liquorice would have to be eaten to have a similar effect in humans and the mechanism of the effect is not understood, this study adds to a growing number of reports that liquorice can trigger potentially dangerous drug interactions.

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Cachexia affects many different chronically ill patient populations, including those with cancer. It results in loss of body weight, particularly of lean body mass (LBM), and is estimated to be responsible for over 20% of all cancer-related deaths. Currently, available drugs are ineffective, and new therapies are urgently needed. The anorexigenic peptide, α-melanocyte stimulating hormone (α-MSH), is believed to be crucially involved in the normal and pathologic regulation of food intake and it was speculated that blockade of its central physiological target, the melanocortin-4 receptor (MC4R), might provide a promising anti-cachexia treatment strategy. The idea is supported by animal studies with agouti-related protein (AgRP), the endogenous inverse agonist at the MC4 receptor, which was found to affect two hallmark features of cachexia: to increase food intake and to reduce energy expenditure.

In 1998, it was reported that MC4R mutations were associated with inherited human obesity. MC4R mutations have a prevalence of 1-2.5% in people with body mass indexes greater than 30, making it the most commonly known genetic defect predisposing people to obesity.

Researchers at Santhera Pharmaceuticals have now published results with orally available MC4R antagonists in an animal model of tumour-induced cachexia. Once daily oral administration of both compounds SNT207858 and SNT207707, starting the day after tumour implantation, significantly reduced the tumour induced weight loss. SNT207707 binds to the MC4 receptor with an affinity of 8 nM and shows a more than 200-fold selectivity vs. MC3 and MC5. SNT207858 is a 22 nM MC4 antagonist with a 170-fold selectivity vs. MC3 and a 40-fold selectivity versus MC5.

Full details of the study are published in the journal PLoS One.

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To grow beyond a certain size, solid tumours need to secure an adequate blood supply and are able to induce angiogenesis to achieve this. Many modulators of angiogenesis have been identified including growth factors such as vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), integrin and cadherin adhesion molecules, and matrix-degrading enzymes. Although angiogenesis inhibitors that target the VEGF pathway such as bevacizumab (Avastin™) and sunitinib (Sutent™) have shown good results in some patients, trials of integrin inhibitors have generally proved less successful.

Integrins are cell surface receptors that play a role in defining cell shape, attachment, and mobility as well as in signal transduction and regulation of the cell cycle. Research funded by Cancer Research UK now suggests that the lack of clinical efficacy of integrin inhibitors may be due to insufficient levels of drug in the body. In laboratory studies, low levels of the experimental integrin inhibitor, cilengitide, were found to boost rather than block tumour cell growth. Cilengitide is a cyclic Arg-Gly-Asp peptide which, at higher doses, binds to and inhibits the activities of the αvβ3 and αvβ5 integrins, thereby inhibiting endothelial cell-cell interactions, endothelial cell-matrix interactions, and angiogenesis. The research, which is published in the journal Nature Medicine, found that low (nM) doses of cilengitide stimulated the supply of blood to the tumour and promoted its growth. Although very low doses of the drug did not have a direct effect on cancer cells, when they were grown alongside blood vessel cells low levels of cilengitide boosted growth of the cancer cells. Low concentrations of cilengitide were shown to promote VEGF-mediated angiogenesis by altering αvβ3 integrin and VEGFR2 trafficking, promoting endothelial cell migration to VEGF.

Even when high doses of cilengitide are administered to patients, blood levels fall in a few hours down to the levels that stimulated rather than blocked tumour growth, suggesting a reason for the poorer than expected efficacy in clinical trials. The study suggests that changing the route of administration or dosing schedule of integrin inhibitors may enhance their clinical efficacy.

Note added 25/3/09, 16.15: Merck Serono released a statement regarding media coverage on pre-clinical integrin inhibitor study in animal model, published in Nature Medicine (22nd March 2009), stating:

Isolated preclinical animal experiment findings do not reflect the actual clinical experience.

The press release can be seen here.

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Cyclosporin A (CsA) and tacrolimus (FK-506) are immunosuppressive drugs used to prevent rejection after allogeneic organ transplants. The drugs bind to specific immunophilins and the resulting complexes (IS-IP complexes) then inhibit calcineurin (CN) and hence production of interleukin-2 (IL-2), a cytokine instrumental in discriminating between self and non-self. Although both CsA and tacrolimus provide benefit in terms of patient and graft survival following transplantation, their potential for treating chronic inflammatory diseases is restricted by adverse side effects.

New research into the mechanism of action of these drugs, led by researchers at the Centro Nacional de Investigaciones Cardiovasculares in Madrid and published in the journal Molecular Cell, however, may offer the promise of safer immunosuppressants.

Previous work had shown that the phosphatase CN regulates a transcription factor known as nuclear factor of activated T cells (NFAT). The physical interaction between CN and NFAT is an essential step in the activation of NFAT-dependent genes by calcium signals, and the new study was designed to investigate how NFAT and IS-IP complexes interact with CN. The research showed that both NFAT and IS-IP complexes compete for the same hydrophobic pocket at the interface of the two calcineurin subunits, and identified small peptides that bind to this site. Identification of compounds that interact specifically with this binding site could lead to the discovery of less toxic immunosuppressants since at least some of the side effects of CsA and tacrolimus are thought to be related to formation of the IS-IP complex and to be independent of CN.

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Results of a phase II clinical trial of a novel oral thrombin receptor antagonist (TRA) were published in the March 14^th issue of The Lancet.

SCH 530348 met the study’s primary endpoints of safety and tolerability, and caused no increase in major and minor Thrombolysis in Myocardial Infarction (TIMI)-scale bleeding when given with current standard antiplatelet therapy (aspirin and clopidogrel) for patients undergoing percutaneous coronary intervention (PCI), commonly known as balloon angioplasty, with stenting. Thrombin is a protein with key roles in blood coagulation – as well as activating platelets for aggregation, thrombin is involved in the production of fibrin, a fibrous protein involved in blood clotting and wound healing. Patients with blocked coronary arteries undergoing PCI are at risk of blood clots and are usually treated with anti-clotting drugs such as aspirin or clopidgrel. Although these drugs are effective at preventing blood clots, they increase the risk of bleeding and there is a need for safer drugs to prevent clots during PCI.

The new study, which involved over 1000 patients, was designed to evaluate the safety and efficacy of SCH 530348 in patients undergoing PCI. SCH-530348 acts as an antagonist at the thrombin receptor and reduces thrombin-induced platelet activation but doesn’t interfere with thrombin’s role in fibrin synthesis. Compared to standard-of-care treatment alone, addition of SCH 530348 led to much greater inhibition of thrombin-receptor agonist peptide (TRAP)-induced platelet aggregation in both loading and maintenance doses.

SCH 530348 is related to the natural product, himbacine, and has a complex structure with seven stereogenic centres but can be synthesised efficiently using a diastereoselective intramolecular Diels-Alder reaction which generates the core tricyclic structure. SCH 530348 is being developed by Schering-Plough for the prevention and treatment of atherothrombotic events in patients with acute coronary syndrome and in those with prior myocardial infarction or stroke, as well as in patients with existing peripheral arterial disease. SCH 530348 is currently being evaluated in two large-scale multinational, randomized, double-blind, placebo-controlled Phase III clinical trials.

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Bacterial resistance – often arising as a result of over, or inappropriate, use of antibiotics – is a major obstacle to the treatment of many bacterial infections. Recently, interference with quorum sensing has emerged as a strategy for the development of new antibiotics which minimises the evolution of drug-resistant strains. Quorum sensing is a process used by bacteria to coordinate gene expression according to local population densities. The bacteria secrete signalling molecules, known as autoinducers, and have receptors that specifically recognize the signalling molecules released by other bacteria of the same or different species. Bacterial cell density and concentration of autoinducers control factors such as expression of virulence factors, pathogenicity and biofilm formation.

Writing in the journal Nature Chemical Biology, researchers from Albert Einstein College of Medicine of Yeshiva University have recently described the effectiveness of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN) inhibitors against Vibrio cholera and Escherichia coli O157:H7. MTAN plays a key role in the synthesis of autoinducers essential for bacterial quorum sensing and the absence of the nucleosidase in mammals suggests that it is likely to be an attractive target for antimicrobial drug design. Three transition state analogue inhibitors of MTAN were found to be highly potent at blocking quorum sensing, bacterial virulence and biofilm formation. Importantly, the effect persisted for several generations.

See also this earlier article on quorum sensing.

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Toxoplasmosis is a parasitic infection caused by the protozoan, Toxoplasma gondii. The parasite is able to infect most warm-blooded animals, including humans, although the primary host is the cat family. Infection is via ingestion of infected meat, cat faeces or through mother-to-child transmission. The majority of infected humans display no symptoms, but those with suppressed immune systems, such as AIDS patients or pregnant women, are at greater risk of developing serious illness. It has been estimated that up to 30% of the World’s population is infected with the parasite.

Following the initial acute phase of infection, a latent phase ensues with the parasite forming cysts in muscle and brain. It is known that infection can modify an animal host’s behaviour and experiments in mice have demonstrated a reduced fear of predators. An increase in levels of the neurotransmitter, dopamine, has been observed in the brains of infected mice. Given the tropism of the parasite for brain tissue, it has long been speculated that Toxoplasmosis in humans may be linked with schizophrenia although no causal relationship has been established.

Scientists at Leeds University have now identified two nearly identical genes in the T. gondii genome with similarity to metazoan tyrosine hydroxylase. Unlike metazoan enzymes of this class, the T. gondii enzymes are bifunctional as they are able to utilize tyrosine and phenylalanine as substrates. Whilst the biological role(s) of a bifunctional tyrosine hydroxylase in T. gondii remain unclear, it is possible that the enzyme is required for supply of tyrosine for protein synthesis. Based on the higher substrate selectivity for tyrosine, however, synthesized tyrosine will be converted to L-DOPA. L-DOPA is the precursor to dopamine and it is plausible that the parasite tyrosine hydroxylase is responsible for the increased dopamine levels detected in the brains of infected rodents.

T. gondii’s ability to synthesise dopamine suggests a potential link with neurological conditions such as Schizophrenia, Parkinson’s disease, Tourette’s syndrome and attention deficit disorders.

The full paper is published in the March 11^th issue of PLoS One.

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NF-κB is a transcription factor that plays a key role in regulating cellular responses to stimuli such as stress and bacterial and viral infections. In un-stimulated cells, NF-κB dimers are sequestered in the cytoplasm by inhibitors known as IκBs (inhibitors of κB) which mask the nuclear localization signals of NF-κB. When the cell is stimulated, IκB proteins are degraded and the NF-κB complex is free to enter the nucleus where it can activate gene expression. The activation of particular genes by NF-κB then leads to a physiological response such as inflammation, an immune response, cell survival or cell proliferation. Once in the nucleus, NF-κB also turns on expression of IκBs, thus forming a feedback loop which regulates activity. Inappropriately regulated NF-κB has been linked to many different types of tumour as well as to inflammatory diseases.

Recent studies have shown that degradation of NF-κB in the nucleus provides an alternative mechanism for regulating its activity and researchers at the University of Illinois have now shown how this process is controlled. They found that TNF-α stimulates methylation of the RelA subunit of NF-κB by lysine methyltransferase Set9 at lysine residues 314 and 315 both in vitro and in vivo. Methylation of RelA inactivates NF-κB by inducing proteasome-mediated degradation of promoter-associated RelA. Depletion of Set9 by siRNA or mutation of the RelA methylation sites was shown to prolong DNA binding of NF-κB and enhance TNF-α-induced expression of NF-κB target genes.

The study, which is published in the journal EMBO, reveals methylation of the RelA subunit of NF-κB as a novel mechanism regulating the turnover of NF-κB and controlling the NF-κB-mediated inflammatory response. The ability to inhibit NF-κB signalling could have applications in the treatment of both cancer and inflammatory diseases.

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