Drug Discovery Opinion

In 1984, shortly after the confirmation of HIV as the causative agent of AIDS, the then United States Health and Human Services Secretary declared that a vaccine would be available within two years. Despite intensive efforts in many laboratories around the world, more than two decades later this ambition has still not been fulfilled.

In 2007, the unexpected and unequivocal failure of one of the largest clinic trials of a vaccine against HIV caused widespread disappointment and dismay but, writing in the journal Nature Medicine, researchers in the United States have now described a vaccination strategy that protects monkeys against infection with the simian immunodeficiency virus (SIV). Instead of trying to stimulate a response from the immune system as with previous vaccines, the new approach delivers genes for antibody-like proteins. The research team first created molecules known as immunoadhesins which bind to SIV and prevent it from infecting cells. After showing that these were effective in cell culture experiments, DNA encoding the SIV-specific immunoadhesins was injected into the monkeys’ muscles using an adeno-associated virus carrier system. The DNA then began to produce immunoadhesins that entered the blood circulation and protected the monkeys from infection. One month after treatment, the majority of the immunised monkeys were protected from infection following challenge with live SIV and all were protected against AIDS. In contrast, unimmunised monkeys all became infected and two thirds died from AIDS complications. High concentrations of the SIV-specific immunoadhesins remained in the immunised monkeys’ blood for over a year. The team hope that long-lasting and complete protection against HIV might also be achieved in humans and are planning initial clinical studies using neutralising antibodies based on those produced by the handful of people who have remained healthy despite long term infection with HIV.

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Floppy Baby Syndrome encompasses a number of incurable genetic diseases that cause severe muscle weakness. In one form of the disease, mutations occur in the ACTA1 gene which encodes the skeletal muscle protein, alpha actin. Most babies born with these mutations are almost completely paralysed and die within their first year but a team of Australian scientists identified a number of less severely affected children who were found to have cardiac actin expressed abnormally in skeletal muscle. In the early foetus, cardiac actin is the most abundant form of the protein in both heart and skeletal muscle but, during development, production of skeletal actin increases until it becomes the dominant form in skeletal muscle. Although the switch occurs in all higher vertebrates, it is not clear why it occurs, or why it occurs only in skeletal muscle.

Mice lacking the ACTA1 gene die within nine days of birth but the team have now shown that if the mice are crossed with transgenic mice expressing ACTC at high levels in skeletal muscle cells the pups survive much longer. ACTC encodes the heart muscle protein, alpha cardiac actin, and almost all of the mice with this gene survived for more than three months and some for more than two years. Although their individual muscle fibres were slightly weaker, their overall muscle strength and locomotive performance were comparable with those of wild type mice. The study demonstrates that cardiac actin is sufficiently similar to skeletal actin to produce adequate muscle function and the team hope that ACTC reactivation might provide an approach for the treatment of diseases caused by ACTA1 mutations. The team are exploring the effects of existing medicines to see whether any of them can ‘switch on’ the ACTC gene in skeletal muscle.

The study is published in the Journal of Cell Biology.

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Although the effects of alcohol have been enjoyed around the world for thousands of years, addiction can lead to significant social and medical problems. Alcohol affects a number of different biochemical pathways and a better understanding of the factors that contribute to alcohol abuse and addiction could lead to improved treatments. Since a lower initial response to the effects of alcohol has been found to correlate with increased risk of future alcoholism, identification of the genes and pathways involved in the acute response might throw light on the genetic factors contributing to addiction. The fruit fly, Drosophila melanogaster, reacts in much the same way as mammals to acute ethanol exposure and a team led by Ulrike Heberlein at the University of California is using the fly to explore the links between genetic make-up and response to alcohol. Their latest find is that flies with a mutant version of a gene that they have designated ‘happyhour’ are less sensitive to the sedative effects of alcohol than flies with a normal copy of the gene. Further studies showed that the epidermal growth factor (EGF)-signalling pathway regulates ethanol sensitivity in Drosophila and that the happyhour protein inhibits this pathway.

The EGF receptor (EGFR) is overexpressed in certain types of carcinoma and drugs that inhibit the EGFR tyrosine kinase such as erlotinib (Tarceva™) and gefitinib (Iressa™) have been successfully developed for the treatment of cancer. The team were able to show that treatment with erlotinib increased acute alcohol sensitivity in both fruit flies and mice and, importantly, also reduced alcohol consumption in a rat model of alcoholism. Although it is not yet clear exactly how alcohol exerts its influence on the EGF pathway or how these changes lead to changes in behaviour after alcohol consumption, the authors suggest that inhibition of the pathway could provide a potential treatment for alcohol addiction. Both erlotinib and gefitinib are well-tolerated and are known to cross the blood-brain barrier. The study is published in the journal Cell.

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Morphine was first extracted from the opium poppy in the early years of the nineteenth century and is still the drug of choice for the treatment of severe acute and chronic pain. Paradoxically, chronic morphine use can lead to reduced analgesia and even increased pain sensitivity. A study by researchers at the Leiden University Medical Center and the City University of New York, published in the June edition of the journal Anesthesiology now provides further insights into why this happens. Initially, engagement of opioid receptors was believed to be critical for opioid-induced hyperalgesia but, more recently, morphine has been shown to increase pain sensitivity in triple knockout mice lacking μ-, κ-, and δ-opioid receptors. Hyperalgesia has also been observed in normal mice during co-administration with the non-selective opioid receptor antagonist, naltrexone. In humans, morphine is metabolised primarily to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). M3G has no analgesic effect and accumulation of M3G has been suggested to underlie morphine hyperalgesia in humans. M6G, on the other hand, has analgesic properties similar to those of morphine although some earlier studies had indicated that it can also increase pain sensitivity in both mice and humans.

In the present study, M6G was shown to increase pain sensitivity in both mice lacking opioid receptors and in mice treated with the opioid receptor blocker, naltrexone. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, was shown to block or reverse the increased pain sensitivity caused by acute injection or continuous infusion of M6G in naltrexone-treated mice. In human volunteers, a single intravenous injection of M6G was found to increase heat pain sensitivity for at least 6 hours and the hyperalgesia was not blocked by simultaneous continuous infusion of the opioid receptor blocker, naloxone.

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Asthma involves an immune response to inhaled allergens and chemical irritants, but the limited efficacy of existing treatments aimed at modifying this response suggest that additional physiological mechanisms may be at work in the disease process. In a report published in the May 19th online Early Edition of PNAS, US researchers have now found that the ion channel, TRPA1, plays a key role in allergic asthma. TRPA1 is a ‘chemosensor’ that is activated by mustard oil as well as by a number of endogenous and exogenous stimuli known to be triggers of asthmatic inflammation. The team found that, compared with wild type mice, animals that did not express TRPA1 showed fewer asthma symptoms, with reduced inflammation, airway mucus and bronchoconstriction. Although the exact role of TRPA1 in asthmatic inflammation is not yet understood, the ion channel is known to be activated by allergens such as cigarette smoke that can trigger asthma attacks. TRPA1 is found in airway nerves and the researchers believe that blocking TRPA1 may prevent infiltration of the lung by the inflammatory cells responsible for asthma symptoms such as wheezing and overproduction of mucus. In further studies, the team went on to show that treatment with the TRPA1 antagonist, HC-030031, reduced the symptoms of allergic asthma in mice. TRPA1 antagonists have previously been shown to reduce chronic inflammatory and neuropathic pain. The discovery of a role for TRPA1 as a neuronal mediator of allergic airway inflammation could lead to new treatments for allergic asthma and Hydra Biosciences, whose scientists contributed to the study, hope to start human clinical trials with a novel TRPA1 inhibitor within 12 months.

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A number of human studies have linked lack of sleep to weight gain – decreased insulin sensitivity and glucose tolerance as well as disruption of the natural balance between the appetite hormones grehlin and leptin have been put forward to explain this link between disrupted sleep and weight gain. In studies in rodents, researchers at Merck have now shown that T-type calcium channels regulate both sleep and body weight maintenance. Mice lacking Ca_V3.1 T-type calcium channels were known to have altered sleep/wake patterns and the new study showed that these mice are also resistant to weight gain induced by a high fat diet.

Writing in the Journal of Clinical Investigation, the researchers report that the knock-out mice gained significantly less weight and had less body fat than their wild-type littermates when fed a high fat diet. The resistance to weight gain of the knock-out mice could not be fully explained by reduced food intake, an overall increase in activity or increased metabolic rate. In further studies, a selective T-type channel antagonist, TTA-A2, was shown to prevent, and even reverse, weight gain induced by a high fat diet, and also to improve body composition to greater extent than the widely used appetite suppressant, fenfluramine. TTA-A2, when dosed either prior to the sleep phase or during the wake phase, was found to promote sleep – a result which was unexpected since the knock-out mice have increased wake time compared with wild type animals. Although the reasons for the observed differences between pharmacological antagonism and genetic knock-out remain to be fully explained, the study highlights the potential for antagonism of T-type calcium channels as a novel weight loss strategy.

The authors suggest that the benefits of T-type calcium channel antagonists may be the result of better alignment of feeding patterns and the circadian rhythm and that sleep or circadian treatments may be of particular benefit for people struggling to lose weight or maintain weight loss because of poor diet.

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Human cytomegalovirus (hCMV) is a widespread member of the herpes family of viruses, with well over half of the world’s adult population thought to be infected. Most individuals are infected in early childhood and remain infected with latent virus for the rest of their lives. For the majority of people, infection is asymptomatic and usually undiagnosed but immunosuppression, caused by HIV-1 infection or drug therapy after transplant surgery, can lead to reactivation of the virus and overt infection. A number of studies have linked hCMV infection to cardiovascular disease, but the mechanisms underlying the pathology are not well understood. Writing in the May 15^th edition of PLoS Pathogens, a team led by researchers at Beth Israel Deaconess Medical Center have now shown that infection with murine CMV (mCMV) leads to a significant increase in arterial pressure in mice. mCMV infection alone was sufficient to cause the observed increase in blood pressure. mCMV infection alone did not cause atherosclerosis in the aorta but, when combined with a high cholesterol diet, did cause classic plaque formation. The team went on to show that mCMV stimulated production of pro-inflammatory cytokines, IL6, TNF-α and MCP-1 which have previously been linked to high blood pressure. They also showed that mCMV infection induced renin expression in an infection dose-related manner in mouse renal cells and that hCMV induced a similar increase in human vascular endothelial cells. In mice, mCMV infection was additionally shown to lead to an increase in angiotensin II levels in serum and in tissue from the aorta.

Since the roles of renin and angiotensin II in hypertension are well established, the study provides a mechanism by which persistent CMV infection might increase blood pressure and also suggests that vaccination or antiviral therapy could have the potential to provide new treatments for cardiovascular disease.

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Two recent reports in the British Journal of Cancer highlight the importance of chemokines in the spread and progression of cancer.

Using a mouse xenograft model, researchers from the University of Nebraska Medical Center showed that CXCR1 and CXCR2 receptors are important in the progression of melanoma, the most aggressive form of skin cancer. The study, which is published in the May 12^th issue of the journal, investigated the effect of overexpression of CXCR1 or CXCR2 in two different human melanoma cell lines – one non-tumourigenic and the other with low tumourigenicity. Both cell lines showed low endogenous expression of the receptors. Both CXCR1- or CXCR2-overexpressing melanoma cells showed enhanced proliferation, chemotaxis and invasiveness in vitro. In vivo, overexpression induced tumourigenicity in the non- tumourigenic cell line and significantly enhanced tumour growth in the low-tumourigenic cell line. Tumours generated from CXCR1- or CXCR2-overexpressing melanoma cells showed significantly increased tumour cell proliferation and microvessel density as well as reduced apoptosis, indicating an essential role for the receptors in growth, survival, motility and invasion of human melanoma.

The second study, published in the 12^th May advance online edition of the journal, investigated the effect of CXCR3 receptors on the metastasis of colorectal cancer (CRC). Liver and lung metastases are the main cause of death from colorectal cancer and the study looked at the ability of CXCR3-expressing CRC cell lines from humans or mice to induce metastases in immunodeficient and immunocompetent mice, respectively. In vitro, activation of CXCR3 on both human and mouse CRC cells by its cognate ligands induced migratory and growth responses which could be blocked by the CXCR3 receptor antagonist, AMG487. In vivo, preventative or curative treatment with AMG487 markedly inhibited implantation and growth of both human and mouse CRC cells in the lung, but had no effect on metastases affecting the liver.

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The axons of nerve cells are sheathed by an insulating layer of myelin which is made up of about 80% lipid and 20% protein. Demyelination, leading to impaired or lost conduction of signals along the nerve, is a hallmark of multiple sclerosis (MS). In relapsing-remitting MS myelin can be replaced but, after repeated attacks, the repair system becomes less efficient. Researchers at the University of Medicine and Dentistry of New Jersey have now identified a key pathway which regulates the production of new oligodendrocytes – the myelin-producing cells of the CNS – and the production of myelin. They found that activation of the mammalian target of rapamycin (mTOR) is essential for oligodendrocyte differentiation at the late progenitor to immature oligodendrocyte transition. The effects were found to be mediated via two distinct signalling complexes, mTORC1 and mTORC2. mTORC2 was found to control myelin gene expression at the mRNA level whereas mTORC1 influenced expression of myelin basic protein via an alternative mechanism.

Although it remains to be determined whether stimulation of the mTOR pathway or removal of some inhibitory mechanism would be most appropriate, allowing the pathway to function normally could provide new treatments for MS and other demyelinating diseases.

The study is published in the May 13th online edition of the Journal of Neuroscience.

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Meningitis – infection of the cerebrospinal fluid and protective membranes surrounding the brain and spinal cord – can be caused by infection with either viruses or bacteria. Viral meningitis is typically relatively mild and self-limiting whereas bacterial meningitis is much more serious and can result in severe brain damage or even death. Bacterial meningitis in children is almost exclusively caused by infection with one of three bacterial strains: Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenza. Exactly how these bacteria are able to breach the blood-brain barrier and cause infection was not understood, but researchers from the University of Nottingham and St. Jude Children’s Research Hospital have now discovered that all three pathogens use the same receptor on human cerebrovascular endothelial cells to begin the process of crossing the barrier. Bacteria need to have some way of fixing their position and becoming established and use attachment molecules – known as adhesins – to achieve this. Some bacteria take the attachment process a stage further and use the adhesins as a first step towards gaining entry into host cells. It was known that the three bacteria responsible for most cases of meningitis share the same strategy for the second step of crossing endothelial cells and the new study has shown that they also use the same host cell receptor, the laminin receptor, for initial attachment. Other infectious agents known to use the laminin receptor to gain access to the CNS include prions and some neurotropic viruses, although the interaction of the laminin receptor with bacteria appears to be somewhat different to that of other pathogens.

The study suggests that blocking the interaction between bacterial adhesins and the laminin receptor might offer broad protection against bacterial meningitis and may lead to better treatments and prevention strategies. The study is published in full in the Journal of Clinical Investigation.

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