Drug Discovery Opinion

Although very different at a molecular level, hepatitis viruses B and C (HBV and HCV) both infect only humans and chimpanzees which means that there is a lack of suitable small animal models for studying viral lifecycles and for testing new drugs. One alternative would be to study the viruses and test new compounds in liver cells grown in vitro, but human hepatocytes are very difficult to grow and maintain in culture.

A team of researchers led by scientists at the Salk Institute has now provided a solution to the problem by generating a mouse with a liver that is almost completely ‘humanised’. The team had previously generated a mouse with a partially humanised liver but wanted to achieve more complete transformation. Around 95% of the liver cells of the new mice are human in origin and the animals are susceptible to infection by both HBV and HCV. Mice infected with HCV were shown to respond to drugs such as pegylated interferon α2a and ribavirin that are used to treat human patients. Adefovir dipivoxil, used to treat HBV patients, was found to lower viral titres in mice infected with HBV.

The mice were generated by using genetic and pharmacological pressures to lead to a growth disadvantage for mouse hepatocytes and positive selection for transplanted human hepatocytes. The mice provide a new way to study pathogens that target the human liver and to test drugs to treat human hepatitis. In the future, the mice could also be used to study other hepatotrophic pathogens such as malaria, as well as cirrhosis and liver cancer.

The study is published in the Journal of Clinical Investigation.

Ion channels, proteins that regulate the transfer of ions across the cell membrane, can be broadly classified according to ‘gating mechanism’ – what makes the channel open and close. Voltage-gated channels respond to a voltage gradient across the plasma membrane whereas ligand-gated channels are activated by binding of extracellular ligands or intracellular second messengers. Recent detailed studies of ion channels are showing, however, that things are not quite so simple.

Researchers in the US and Germany have now shown that they can confer significant voltage dependence to the inwardly rectifying K⁺ channel, K_ir6.2, by introducing a point mutation, L157E. K_ir6.2 is a ligand-gated channel that lacks a canonical voltage-sensing domain (VSD). In classical models of voltage-dependent gating, the VSD strongly influences opening and closing of the pore-forming domain so that the channel open probability is reduced to virtually zero at sufficiently negative voltages and increased to near unity upon depolarization. Previous observations have shown that such ‘tight coupling’ between the VSD and the pore does not apply to all channel types and the new study shows that substitution of charged residues at pore-lining positions can affect channel gating in very unexpected ways. Comparing K_ir6.2[L157E] with wild type K_ir6.2, the team found that the probability of opening under conditions of low intracellular K⁺ was much greater for the mutant channel. The presence of a natural ligand, phosphatidyl inositol bis-phosphate (PIP₂), removed the voltage dependence of the mutant channel. Both voltage- and ligand-dependent gating of K_ir6.2[L157E] were highly sensitive to intracellular [K⁺], indicating an interaction between ion permeation and gating.

The authors of the study, which is published in PLoS Biology, propose that ions flowing through the ion channel pore can significantly affect channel activity and that the mechanisms of voltage-gating and ligand-gating may be more closely linked than previously supposed. They further suggest that such interactions are likely to be a general, if latent, feature of the superfamily of cation channels.

Some 200 million people worldwide are estimated to be infected with the hepatitis C virus (HCV) which can eventually lead to cirrhosis, liver cancer, and in some cases, death. The current standard-of-care treatment for persistent infection – a combination of pegylated interferon-α and ribavirin – is able to clear the infection in around 50% of patients. In some patients, however, treatment is associated with haemolytic anaemia which may be severe enough to need dosage reduction or even discontinuation of treatment.

A team led by scientists at Duke University’s Institute for Genome Sciences & Policy (IGSP) have now discovered that loss of function mutations in the gene ITPA, which encodes the enzyme inosine triphosphatase, protect against the development of anaemia. Previous studies had identified the genetic variants with enzyme deficiency and, through a genome-wide association study, the Duke team were able to show that they were also protective against anaemia caused by ribavirin. The finding may offer new treatment opportunities for HCV patients with coronary artery disease or kidney disease who are often not treated with ribavirin because of fears that anaemia could exacerbate their condition.

Inosine triphosphatase deficiency was first recognised over 30 years ago and is not thought to be clinically important. A diagnostic test that could predict deficiency, and hence reduced susceptibility to ribavirin-associated anaemia, would allow broader treatment options for HCV patients.

The study is published in the journal Nature.

Compared with the genome of a normal cell, that of a cancer cell has sections that are missing, repeated or scrambled. Although some of the mutations are probably driving the cancer, others may have little relevance to the cancer’s progression. To understand which mutations are important in the development and spread of cancer, a multi-national team including researchers from the Broad Institute and the Dana-Farber Cancer Institute have studied over 3000 primary human cancer specimens representing more than 26 cancer types, including lung, prostate, breast, ovarian, colon, oesophageal, liver, brain, and blood cancers.

Classically, cancers have been classified (and treated) by their tissue of origin but, more recently, it has been realised that oncogenes are not restricted to one type of cancer. In the new study, the team found that amplifications and deletions of regions of the genome, known as somatic copy-number alterations or SCNAs, are not evenly distributed across the cancer genome but are concentrated in less gene-rich regions where they may be better tolerated. As with other mutations seen in cancer cells, many SCNAs may not play an important role in cancer growth but some will identify genes that help to promote and maintain cancers.

The most common SCNAs were found to be either relatively long (the length of an entire chromosome or a single chromosome arm) or short (about 1.8 million base pairs). More than 150 regions of short, or focal, SCNA – which because of their compact size are more likely to pinpoint important cancer-linked genes – were found to be altered at significant frequency across several cancer types. Of these, 122 did not coincide with genes already known to be amplified or deleted in cancer but the team also found copy number changes in BCL2 family genes – which are already linked to cancer and are the target of some cancer drugs – in over half of all samples. Most focal SCNAs were not unique to one type of cancer suggesting that common genetic mutations underlie different types of tumour, a finding which may lead to improved diagnosis and more targeted treatments for cancer.

The study is published in the journal Nature and the researchers have also built an online tool, the Tumorscape portal, to allow other researchers to freely access their data.

Although the disorder is not very well known, narcolepsy is thought to affect 1 in 2000 individuals and this figure may be higher as a consequence of under-reporting and under-diagnosis. The most common symptom is excessive daytime sleepiness (EDS), which may be accompanied by sudden loss of muscular control (cataplexy) triggered by strong emotions. Narcoleptics may also experience sleep paralysis (short periods of paralysis when waking or falling asleep), hypnagogic or hypnopompic hallucinations (vivid images or sounds, respectively, when waking or falling asleep) or automatic behaviour (when routine activities are continued during a sleep episode).

For the last ten years it has been known that narcoleptics have a deficiency in hypocretin (orexin), a neurotransmitter involved in control of sleep/wakefulness. In parallel with the neurotransmitter deficiency there is a massive loss of hypothalamic neurons that produce hypocretin and it has been hypothesised that this results from an autoimmune response.

Swiss scientists have now identified autoantibodies to Tribbles homolog 2 (Trib2), an autoantigen previously identified in autoimmune uveitis, in narcolepsy patients. The team developed a transgenic mouse model to identify peptides enriched within hypocretin-producing neurons that could serve as potential autoimmune targets. Having identified enrichment of Trib2 in the mouse hypocretin neurons, the team went on to analyse sera from narcoleptics. Narcolepsy patients with cataplexy had higher Trib2-specific antibody titres compared with either normal controls or patients with other inflammatory neurological disorders. Trib2-specific antibody titres were highest early after narcolepsy onset, sharply decreased within 2–3 years, and then stabilized at levels substantially higher than that of controls for up to 30 years. Additionally, high Trib2-specific antibody titres correlated with the severity of cataplexy.

The study, published in the Journal of Clinical Investigation, provides the first evidence that narcolepsy is an autoimmune disorder.

Several million people in Latin America are estimated to have Chagas disease, although most will not know that they are infected. The disease is caused by the protozoan Tryptanosoma cruzi and is transmitted by the bite of the blood-sucking beetles known as ‘kissing bugs’ because of their tendency to bite close to the mouth. Although the disease typically produces very mild symptoms in the early stages, chronic disease can lead to severe damage to the heart and GI tract and is often fatal. Severe disease and death occur many years after initial infection – even when the parasites are no longer present – and Brazilian scientists speculated that T. cruzi DNA might be retained in the body and that the resulting genomic alterations could explain the rejection of heart tissue by the host immune system. An unusual feature of virulent T. cruzi is that extra-nuclear mitochondrial DNA known as the kinetoplast (kDNA) accounts for 15-30% of the total cellular DNA. The kDNA is made up of thousands of minicircles which can insert into the genome of an infected individual. Depending on where the parasitic DNA is inserted, it can affect genes involved in cell communication, correct function of the immune system and even our sense of smell. The parasitic DNA can also be incorporated into the DNA of sperm and eggs and so can appear in the genome of children who have never themselves been in direct contact with the parasite.

Although it has been known for some time that the human genome contains significant amounts of viral DNA that have evolved with us over millions of years, the new research suggests that the transfer of mitochondrial DNA from T. cruzi to the human population may also be contributing to human genetic diversity. The parasite is especially well placed to fulfil this role since it infects millions of people, many of whom will migrate to other continents, infects many species of wild animals, and rarely kills its human victims below the age of forty.

The study is published in PLoS ONE.

Around 85% of all tumours are epithelial in origin and have epithelial markers on the cell surfaces. In breast, ovarian, pancreatic and colon-rectal cancers, metastasis – when cells break away from the primary tumour to initiate a new tumour elsewhere in the body – is often fatal. A process known as epithelial-mesenchymal transdifferentiation (EMT) plays a role in releasing epithelial cells from the surface of solid tumours and transforming them into transient mesenchymal cells which have reduced cell-cell adhesion properties and increased ability to migrate and establish new tumours. It was already known that transforming growth factor-β (TGF-β) induces EMT but the downstream events were unclear.

Scientists in the US have now shown that TGF-β triggers the formation of a protein known as disabled-2 (Dab2) which, in turn, activates the EMT process. An increased understanding of the signalling pathway for modulating EMT could lead to the design of drugs to delay or halt EMT and control tumour metastasis. The discovery may also help to understand the progression of other diseases.

The study is published in Nature Cell Biology.

Protein kinases play important roles in regulating most cellular processes and are commonly activated in cancer cells. A number of kinase inhibitors – including antibodies and small molecules –have already been approved for the treatment of cancer and many others are currently being tested. The majority of kinase inhibitors developed so far are ATP mimetics identified by high-throughput screening of catalytic kinase domains at low ATP concentration. Such compounds – so-called type I inhibitors – may lack specificity for individual kinases and/or be less effective when ATP concentrations are high. Crystal structures have revealed that some compounds – the type II inhibitors – occupy an allosteric site accessible only in the inactive conformation of the kinase and researchers at the Moores Cancer Center at the University of California have now designed selective type II inhibitors of PDGFRβ (important for pericyte recruitment) and B-RAF (important for endothelial cell survival).

Using the X-ray crystallographic structure of the type II inhibitor, sorafenib, bound to B-RAF, the team designed a small library of compounds based on a constrained amino-triazole scaffold predicted to stabilise kinases in the inactive state. The compounds were then tested for antivascular activity in both cell-based models and a zebrafish embryogenesis model. Compound 6 was found to inhibit both PDGFRβ and B-RAF cellular signalling – which produces a synergistic effect on tumour growth – but to have no effect on a variety of other cellular targets. The compound showed antiangiogenic activity in both zebrafish and murine models of angiogenesis and was also shown to suppress murine orthotopic tumors in both the kidney and pancreas.

The study is published in the Proceedings of the National Academy of Sciences.

Cystic fibrosis (CF) results from a genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) that results in impaired transport of chloride and bicarbonate ions. Patients with CF have thickened mucus, accompanied by inflammation, which affects the lungs and organs of the intestinal tract. Although the disease has received much scientific attention, current treatments only manage the symptoms and affected individuals continue to suffer from reduced life expectancy.

A new study from researchers at University of California, San Diego School of Medicine, has now identified defects in signalling mediated by peroxisome proliferator-activated receptor-γ (PPAR-γ) that contribute to disease symptoms. Examining colonic epithelial cells and whole lung tissue from CFTR-deficient mice, the team found reduced expression of genes that are normally activated by PPAR-γ. Lipidomic analysis of the colonic epithelial cells suggested that the defect resulted in part from reduced amounts of the endogenous PPAR-γ ligand, 15-keto-prostaglandin E₂ (15-keto-PGE₂). The researchers were able to partially restore gene expression by treating the mice with rosiglitazone, a PPAR-γ agonist used in the treatment of diabetes, reducing the severity of disease.

Rosiglitazone had no effect on chloride secretion in the colon, but increased expression of carbonic anhydrases 2 and 4 (Car2 and Car4) resulting in increased bicarbonate secretion and reduced mucus retention.

The study, published in Nature Medicine, suggests that levels of 15-keto-PGE₂ could provide a marker for patients who might benefit from treatment with a PPAR-γ agonist.

Stuttering or stammering affects people of all ages and races and most often starts between the ages of two and five, when children start to develop language skills. Although most children outgrow stuttering, about one percent of adults continue to stutter. The underlying causes are not well understood but stressful situations can make stuttering more severe and, especially in the past, the condition was believed to have a social or emotional component. It has now been recognised that developmental stuttering runs in families and a section of chromosome 12 has been linked to stuttering in a group of Pakistani families. In a study published in the New England Journal of Medicine, researchers at the National Institute on Deafness and Other Communication Disorders (NIDCD) have now refined the location on chromosome 12 to mutations in three genes, GNPTAB, GNPTG and NAGPA, which encode proteins involved in lysosomal recycling of unwanted cellular components. Mutations in two of these genes had previously been linked with rare, and often fatal, lysosomal storage disorders.

Although only a small proportion of stutterers are likely to have these genetic mutations, the study is the first to pinpoint specific gene mutations as a potential cause of stuttering. Recently, enzyme replacement therapy has been developed to treat lysosomal storage disorders and, if the mutations involved in stuttering also prove to involve loss of enzyme function, such treatments could eventually also be effective for this group of stutterers.