MicroRNAs (miRNAs) are small (21-23 nucleotides in length) single stranded RNA molecules which are not translated into proteins but whose main function is to regulate gene expression. Almost all types of tumour have abnormal – usually reduced – miRNA expression, and scientists at Johns Hopkins Medical School, Nationwide Children’s Hospital, and Ohio State University have now shown that replacing missing miRNAs in mice with liver cancer can rapidly kill the tumour cells whilst leaving healthy cells untouched. The team found that hepatocellular carcinoma (HCC) cells had reduced levels of miR-26a, a miRNA that is normally expressed at high levels in a variety of tissues. In vitro, expression of miR-26a in liver cancer cells was shown to cause cell-cycle arrest associated with direct targeting of cyclins D2 and E2. Systemic administration of miR-26a to mice with HCC using an adeno-associated virus vector inhibited the proliferation of the cancer cells and protected the animals from disease progression. Normal liver cells were unaffected by the treatment. After three weeks, 8 out of 10 mice treated with the miRNA showed only small tumours or a complete absence of tumours, whereas 6 out of 8 sham-treated mice experienced aggressive disease progression.

The study shows that liver cancer can be successfully treated in an animal model of disease by replacing ‘missing’ miRNA using a delivery vector that is suitable for use in the clinic. Although more work is needed before the technique could be used in patients, it could provide an effective and safe treatment for human HCCs, which account for 80 – 90% of all liver cancers and generally have a poor prognosis. If suitable delivery systems can be developed, the technique could also be used to treat other diseases caused by ‘missing’ miRNA.

The study is published in the June 12^th edition of Cell.

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Huntington’s disease (HD) is an inherited neurological disorder that causes a wide range of symptoms including involuntary movements, clumsiness, lack of concentration, memory lapses, mood swings, and depression. Although the disease mechanism is not fully understood, it is known that sufferers have a defect on the short arm of chromosome 4 and produce abnormal versions of the protein, huntingtin. One of the puzzles surrounding the progression of HD has been that, although abnormal huntingtin proteins accumulate in cells everywhere in the body, they predominantly kills cells in the corpus striatum, the part of the brain that controls movement.

Writing in the journal Science, scientists at John Hopkins School of Medicine now report that the answer may lie with the small G protein, Rhes (Ras homologue enriched in striatum), which is found almost exclusively in the corpus striatum. In cell culture experiments using both mouse and human cells, Rhes was found to bind much more tightly to mutant huntingtin than to normal protein. When added singly to cell cultures, neither Rhes nor mutant huntingtin had any effect on survival but, when they were added together, half of the cells died within 48 hours.

Abnormal huntingtin proteins aggregate and form clumps, but there are fewer of these clumps in the corpus striatum of HD patients than in other brain regions or elsewhere in the body, suggesting that clumping of the protein may actually protect the cells. The team found that, in their cell culture experiments, adding Rhes to cells with abnormal huntingtin led to fewer clumps, although the cells died. The results suggest clumping of abnormal huntingtin may prevent it from causing cell death and that Rhes might be responsible for preventing abnormal protein from clumping. Rhes was also shown to promote sumoylation of mutant huntingtin. The team are currently exploring whether removing Rhes from mice with HD will prevent cell death, and hope that it may eventually be possible to design drugs which will specifically target Rhes to treat HD.

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Toxoplasmosis is a parasitic disease caused by infection with the protozoan, Toxoplasma gondii, whose primary hosts are members of the cat family. Infection of humans usually results from consumption of contaminated water, unwashed vegetables or undercooked meat, or from contact with cat faeces. The incidence of human infection varies widely in different countries and, in both acute and latent infections, symptoms are typically mild and often go unnoticed. The latent phase in humans has, however, been associated with behavioural changes and neurological disorders, including increased reaction times. Recent studies have suggested a protective effect of the rhesus factor (RhD antigen) against the negative effects of latent T. gondii infection on reaction times, especially in heterozygous individuals. Uninfected RhD negative men have faster reaction times than uninfected RhD positive men, but heterozygous men with both positive and negative alleles are protected from prolongation of reaction times caused by T. gondii infection.

A study published in the journal BMC Infectious Diseases now suggests that drivers who are rhesus factor (RhD antigen) negative and infected with T. gondii are more likely to be involved in road traffic accidents. Although this is not the first time that an association between toxoplasmosis and driving impairment has been suggested, it is the first prospective study to examine the link, and to consider the effect of RhD phenotype. Almost 4000 Czech male military drivers were tested for T. gondii infection and RhD phenotype at the beginning of a 12-18 months period of compulsory military service. The men were not told whether they were infected or not and were also not told that the incidence of traffic accidents would be monitored. By correlating infection data and RhD phenotype with subsequent traffic accidents reports from military police records, the authors confirmed an increased risk of accidents in infected men, and also demonstrated a protective effect of being RhD positive against this risk. When corrected for factors such as the age of the driver and date of military service, the probability of a traffic accident was found to be higher for RhD negative drivers with high titres of antibodies against T. gondii than for uninfected drivers or for drivers who were RhD positive. Whilst recognising the absence of genotypic data as a limitation of the present study – earlier studies had shown that protection against the deleterious effects of T. gondii infection is largely restricted to heterozygotes – the authors suggest that RhD negative individuals with occupations that need fast reaction times, such as pilots and air traffic controllers, should be regularly tested for infection with T. gondii.

The RhD protein, found on the surface of red blood cells in around 85% of North Americans and Europeans, is highly immunogenic and used to be responsible for a complication of pregnancy. If an RhD negative woman with RhD antibodies – as a result of a blood transfusion or earlier pregnancy –is carrying a baby which is RhD positive as a result of a gene inherited from the father, maternal antibodies will mount an immune response against the red blood cells of the baby. Structural homology data suggest that the RhD protein may act as an ion pump although its function is largely unknown. The authors suggest that the incidence of RhD alleles in human populations may be linked to the advantages conferred to heterozygotes in the presence of endemic T. gondii.

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Cancer stem cells are slowly dividing tumourigenic cells that possess characteristics of normal stem cells. It has been proposed that these cells persist in treated tumours and are responsible for tumour re-growth and metastasis. As a consequence, more effective chemotherapy may be achieved by targeting these cells in addition to the rapidly proliferating tumour cells. So far, the difficulty has been the lack of understanding of cancer stem cells and how they might be selectively targeted over normal stem cells.

In research carried out at The Jackson Laboratory in mice, Alox5 (5-Lipoxygenase) has been shown to be essential for development and maintenance of cancer stem cells in bcr-abl dependent chronic myeloid leukemia (CML). CML did not develop in mice without the Alox5 gene as a result of impaired function of the leukemia stem cells. Importantly, the lack of Alox5 did not affect normal stem cells, indicating distinct pathways in normal and leukemic stem cells for differentiation and self-renewal.

Alox5 is known to be essential for processing fatty acids to leukotrienes, key components of the inflammatory response, and inhibitors of Alox5 have been developed for the treatment of asthma. Using the approved (for asthma) Alox5 inhibitor, Zileuton, the scientists at The Jackson Laboratory were able to demonstrate a greater therapeutic effect in the CML model than the gold standard, Gleevec. Combining the two therapeutics provided an even better response.

The full study is published in the journal Nature Genetics.

Alox5 has also been shown to be over-expressed in certain cancers, for example in colon cancer. The availability of a clinically approved Alox5 inhibitor should enable a rapid progression to clinical studies in cancer.

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Although highly active antiretroviral therapy (HAART) has proved effective at controlling HIV-1 replication, eradication of the virus from an infected individual cannot be achieved with current treatments. Latently infected, resting memory CD4⁺ T-cells and macrophages act as reservoirs for the virus and so called “shock and kill” strategies (activation of HIV-1 in the presence of HAART to block viral spread followed by stimuli leading to cell death) have been proposed to eradicate the virus. Histone deacetylase inhibitors (HDACIs) have been shown to be effective for the “shock” phase, but their use is limited by toxicity at the levels needed for viral reactivation. A team of Italian researchers looked for ways to improve the effectiveness of HDACIs and found that adding buthionine sulfoximine (BSO) to class I HDACIs reduced the concentration of HDACI needed to reactivate HIV-1 in cell culture experiments. The team hypothesised that, since HIV-1 transcription is enhanced by oxidative stress, glutathione depletion might create an intracellular environment that facilitates HIV-1 activation by HDACIs. BSO inhibits γ-glutamylcysteine synthetase, the rate limiting enzyme in glutathione synthesis, and has been studied as an adjunct to chemotherapy. At non-toxic concentrations, class I HDACIs only reactivated virus in some of the cells but, when BSO was added, all of the cells responded and were killed. MS-275, a class I HDACI currently undergoing clinical trials for the treatment of cancer, was one of the compounds found to reactivate HIV-1 more effectively in the presence of BSO. The combination showed low toxicity in uninfected cells.

Although much more work needs to be done, the authors hope that their research may open up new avenues in the search for ways to completely eliminate HIV-1-infected cells from the body.

The study is published in the journal Retrovirology.

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A major challenge in treating autoimmune disease is to prevent tissue damage without generalised immunosuppression. US researchers now suggest that selective damping down of the T_H17 response using compounds such as halofuginone may provide an answer to this challenge.

Writing in the June 5^th edition of the journal Science, they show that halofuginone specifically inhibits the development of T_H17 cells which are believed to play a key role in tissue injury in autoimmune diseases such as inflammatory bowel disease, multiple sclerosis, type 1 diabetes, eczema and psoriasis. When halofuginone was added to cultures of naïve mouse CD4⁺ T-cells containing cytokines that would normally induce differentiation into T_H17 cells, the number of T_H17 cells – but not T_H1, T_H2 or T regulatory cells – was substantially reduced. In cultured human CD4⁺ T-cells, halofuginone also selectively suppressed levels of IL-17, the main cytokine produced by T_H17 cells. In mice with experimental autoimmune encephalitis (EAE), an artificially-induced immune disease resembling multiple sclerosis and marked by infiltration of T_H17 cells into the central nervous system, treatment with low doses of halofuginone significantly reduced both the development of EAE and its severity.

To understand how halofuginone works, the researchers looked at alterations in gene expression in response to drug treatment and found that a cytoprotective signalling pathway, the amino acid starvation response (AAR), was activated. Inhibition of T_H17 differentiation by halofuginone could be overcome by the addition of excess amino acids and was mimicked by AAR activation in response to selective amino acid depletion.

Halofuginone is a synthetic analogue of febrifugine, the active principal of the Chinese herb, chang shan (Dichroa febrifuga), which has been used to treat fever and malaria for more than 2000 years. Febrifugine itself causes severe emesis and gastrointestinal irritation and, in the 1960s, a number of analogues – including halofuginone – were synthesized by U.S. Army scientists looking for novel antimalarials. Halofuginone also inhibits synthesis of collagenase and collagen type 1 and underwent clinical trials for the treatment of scleroderma, a chronic, autoimmune condition of the connective tissue. In animal husbandry, halofuginone (as Stenerol®) is used prophylactically to control coccidial infection in poultry flocks.

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Concert Pharmaceuticals and GlaxoSmithKline recently announced a collaboration to develop deuterium-containing medicines, including CTP-518, a partially deuterated version of the HIV protease inhibitor, atazanavir (Reyataz™), marketed by Bristol-Myers Squibb. Reyataz™ is used in combination therapy to treat HIV/AIDS and, for most patients, the recommended dose is one 300mg tablet daily taken with ritonavir (Norvir™). Ritonavir was originally developed as a ‘stand-alone’ HIV protease inhibitor but is now primarily used, not for its antiviral activity, but to ‘boost’ levels of other protease inhibitors by inhibiting their metabolism. Despite its marked benefits as part of combination therapy, ritonavir is poorly tolerated by some patients and also influences the metabolism of concurrently administered drugs, especially those metabolised by CYP 3A4.

Concert is pioneering the modification of existing medicines by selectively replacing hydrogen atoms with deuterium atoms in the expectation that the modified compounds will have similar activity at the target enzyme or receptor, together with improved ADME properties. CTP-518 has been shown to have similar antiviral potency to atazanavir but slower hepatic metabolism, leading to the hope that it could be used clinically without the need for ‘boosting’ by ritonavir. This could lead to better safety and tolerability for patients and also allow for the inclusion of CTP-518 in fixed dose regimens. CTP-518 is expected to enter Phase I clinical trials in the second half of 2009.

Concert has filed a patent application (WO20081566632) claiming derivatives of atazanavir, including compounds 120 and 122.

The antiviral activities of compounds 120 and 122 were shown to be similar to, or slightly better than, that of atazanavir, both in the presence and absence of serum proteins. In human liver microsomes, compounds 120 and 122 showed an approximately 50% increase in half life compared with atazanavir. Following oral co-dosing in rats, compound 122 showed a 43% increase in half life, a 67% increase in Cmax and an 81% increase in AUC compared with atazanavir. When administered to chimps, both compounds showed around 50% increases in half life compared with atazanavir and about 2-fold increases in urine concentration.

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For the most part, cancer therapy has been aimed at exploiting pathways that are present in cancer cells and not in normal cells but two studies published in the May 29^th issue of the journal Cell suggest potential for an alternative approach. Blocking the activity of oncogenic protein kinases – either with antibodies or small molecules – has become an important field of interest in cancer research but, despite the prevalence of RAS mutations in human tumours, inhibition of oncogenic RAS has not been realised as a therapeutic strategy. As an alternative to directly targeting RAS, US researchers have now identified ‘normal’ genes that are needed for cell survival in the presence of mutant, but not wild-type, KRAS.

To identify genes that are essential for survival only in the context of mutant KRAS, the researchers used a short hairpin RNA (shRNA) library to carry out high-throughput loss-of-function RNA interference (RNAi) screens in cancer cell lines as well as in normal cells. Dozens of potential drug targets were identified including serine/threonine kinase 33 (STK33) and mitotic polo-like kinase 1 (PLK1).

Patients with KRAS tumours are more likely to survive if they also have reduced expression of genes in the PLK1 pathway, suggesting that PLK1 inhibitors may have the potential to prolong survival. Although not required by normal cells, STK33 was found to be essential for the survival of cancer cells, irrespective of their tissue of origin, again suggesting therapeutic potential for inhibitors.

As well as identifying new targets for which it may be possible to develop therapeutically useful inhibitors, the two studies demonstrate the potential of RNAi screens to discover functional dependencies between oncogenes and normal genes in cancer cells. Targeting proteins which are essential for the survival of cancer cells, but not normal cells, could lead to a substantial therapeutic window, especially if only partial knock-down is needed to kill cancer cells.

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The neurodegenerative disorder, Huntington’s Disease (HD, Huntington’s Chorea), is caused by mutations in the gene for the protein Huntingtin (Htt). Mutant Htt (mHtt) results when the number of trinucleotide repeats, in this case the CAG sequence encoding glutamine, exceeds a threshold value. Typically, HD affects patients when the number of repeats is greater than 35.

Although the mechanisms by which mHtt causes the disease are poorly understood, proteolysis of the mutant protein is key and neurotoxicity is attributed to the cleaved N-terminal fragments of mHtt. Scientists at the California Institute of Technology and the Wallenberg Neuroscience Center, Lund, Sweden have now reported that the pro-inflammatory kinase, IKKβ , can regulate mHtt proteolysis in response to neuronal DNA damage.

The team demonstrated that DNA damage in neurons induced by etoposide or γ-irradiation results in cleavage of both wild-type and mutant Htt and that the proteolysis requires IKKβ. Elevation of IKKα, inhibition of IKKβ expression or inhibition of IKKβ catalytic activity all suppressed proteolysis and increased neuronal resistance to DNA damage.

Since elevated neuronal DNA damage is observed in HD patients and animal models of HD, inhibition of IKKβ activity may represent a treatment option for the disease. The study is published in the journal PLoS one.

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Increasingly, scientists and doctors are trying to personalise cancer treatment by identifying genetic alterations involved in the disease in individual patients. One example is the development of a monoclonal antibody, trastuzumab (Herceptin™), directed against the ErbB-2 (HER2) protein which is overexpressed in a subset of breast cancers and leads to a highly aggressive form of the disease.

DNA microarrays have been widely used to study gene expression in cancer and a team of US researchers have now used the technique to search for other genes that are overexpressed in breast cancers. Writing in the journal PNAS, the team describe a meta analysis of 31 breast cancer gene profiling studies comprising almost 3,200 microarrays. As well as correctly identifying the known breast cancer-associated gene, ErbB-2, the study found that the angiotensin II receptor type I (AT₁) was markedly overexpressed (up to 100-fold) in 10-20% of tumours across multiple independent cohorts. AT₁ overexpression was found only in oestrogen receptor (ER)-positive tumours and was also restricted to samples that did not overexpress ErbB-2. In primary mammary epithelial cells, ectopic overexpression of AT₁ together with angiotensin II stimulation led to a highly invasive phenotype that was attenuated by the AT₁ antagonist, losartan. In mice, treatment with losartan (90 mg/kg/day) for eight weeks was found to reduce tumour growth by 30% in AT₁-positive breast cancer xenografts.

AT₁ has previously been linked to cancer and cancer-related signalling pathways and the new study suggests that women with AT₁-overexpressing breast cancer may benefit from treatment with an AT₁ antagonist. Earlier studies have linked polymorphisms in the angiotensin pathway with breast cancer incidence and, although studies have not found a significant relationship between antihypertensive therapy and breast cancer incidence, the authors believe that the studies may not have been sufficiently powered to detect the small changes in incidence that might be expected from a subset of only 10-20% of AT₁-positive patients.

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