News – Page 9 – Drug Discovery Opinion

Mice Can Do What Poppies Can

poppies and mouse — *Papaver somniferum*: Flickr - Alastair Rae; Mouse: Flickr - Sergey Yeliseev

Preparations from the opium poppy, papaver somniferum, have been used for thousands of years to relieve pain. In the early 1800s, Sertürner isolated one of the constituent alkaloids, morphine, which was later shown to be almost entirely responsible for the analgesic activity of crude opium extracts. Since then, morphine and other opiates have been shown to bind to specific receptors, leading to speculation that mammals may be able to biosynthesise morphine. It has also been recognised that humans excrete small but steady amounts of morphine in their urine, but it has been unclear whether this comes from dietary sources or whether the presence of morphine in urine provides evidence that people can actually biosynthesise morphine. The discovery of the simple isoquinoline alkaloid, tetrahydropapaveroline (THP), which can be formed from catecholamines, in both human and rodent brain and in human urine also suggested a possible biosynthetic linkage.

A team led by Nadja Grobe from the Donald Danforth Plant Science Center and Marc Lamshöft from the Institute of Environmental Research, University of Technology Dortmund have now shown conclusively for the first time that mammals are able to synthesise morphine. When either unlabelled or deuterated THP, a potential precursor of morphine, was administered to mice, analysis of their urine showed that the compound had been extensively metabolised. Salutaridine, a known biosynthetic precursor of morphine in the opium poppy, was found amongst the metabolites and when deuterated salutaridinol, the biosynthetic reduction product of salutaridine, was administered to the mice, it was shown to be converted to deuterated thebaine, which was excreted in their urine. Deuterated thebaine was also administered and deuterated morphine together with the related compounds, codeine and oripavine, was recovered in urine.

The study, which is published in the journal PNAS, provides the first evidence that mammals have the capacity to convert THP, known to be present in the brain, into morphine. Although the pathway has now been shown to exist, it is still not clear whether mammals naturally produce morphine as an analgesic.

Strengthening the Case Against Astrocytes in Epilepsy

Epileptic seizures are caused by sudden bursts of excess electrical activity in the brain, leading to a temporary breakdown in normal communication between brain cells. Although it was originally believed that neurones were solely responsible for signalling in the nervous system, it has become increasingly clear that non-neuronal cells called glia – which provide structural and nutritional support for neurones – are also able to modulate neurotransmission. Star-shaped glia known as astrocytes have increasingly been recognised to play a key role in the excessive neuronal synchrony that occurs in epilepsy and researchers at Tufts University and the Children’s Hospital of Philadelphia have now added strong evidence to the case against astrocytes.

The team focussed on reactive astrocytosis, a condition which occurs prominently in response to CNS injury or disease and which has, so far, been difficult to study. Using a virus to selectively cause reactive astrocytosis in mice without triggering broader inflammation and brain injury, the researchers were able to study how the altered astrocytes affected specific synapses in neurones in brain slices from the animals. Normally, neurotransmission is a delicate balance between excitation and inhibition, with the astrocytic enzyme, glutamine synthetase, playing a key role in regulating this balance. In reactive astrocytosis, the astrocytes produce less glutamine synthetase which, in turn, decreases inhibition and leads to the uncontrolled signalling characteristic of epileptic seizures. By adding glutamine – which is depleted as a result of reduced glutamine synthetase activity – the researchers were able to dampen neuronal excitability in the brain slices. The team are continuing to investigate how their research may contribute to developing new treatments for epilepsy and other neurological disorders as well as stroke and traumatic brain injury.

The study is published in the journal Nature Neuroscience.

HCV NS5A Inhibitor Shows Early Promise in Clinic

Hepatitis C virus (HCV) is a leading cause of liver disease and the current ‘standard of care’ treatment, a combination of pegylated interferon-α and ribavirin, is expensive, can cause serious side effects and is only effective for some patients. There are at least 6 known HCV genotypes, with several subtypes and quasispecies within each genotype and these all respond differently to treatment – the combination of pegylated interferon-α and ribavirin is able to clear the virus from about half of patients infected with genotype 1 HCV. In the search for more effective treatments, a number of groups are developing compounds that specifically target HCV. Inhibitors of the viral NS3 serine protease and the NS5B4 RNA-dependent RNA polymerase have already entered the clinic and researchers at Bristol-Myers Squibb have now reported that a compound with a new mechanism of action, BMS-790052, led to dramatic reductions in viral load and produced few side effects in a phase I clinical study. BMS-790052 targets NS5A, an HCV non-structural protein that possesses no enzymatic activity and is thought to regulate viral replication and host cell interactions. The compound, which was identified using a chemical genetics strategy, is active at picomolar concentrations in vitro towards replicons expressing a broad range of HCV genotypes and acts additively to synergistically with interferon and other small molecule antiviral compounds.

The study, which is published in the journal Nature, provides the first clinical evidence that targeting NS5A may be an effective strategy for treating patients who are chronically infected with HCV. Future treatments for HCV infection may involve ‘cocktails’ of drugs that target specific stages in the viral lifecycle, just as HIV infection is managed now, although whether it will be possible to drop interferon from HCV treatment combinations remains to be seen.

First Small Molecules Active Against Ricin, Shiga Toxin

Castor Oil Seeds — Seeds of the castor oil plant, *Ricinus communis*

Ricin, one of the world’s deadliest toxins, was infamously used to assassinate the Bulgarian dissident writer Georgi Markov in 1978 near Waterloo Bridge in London. Markov died a few days after a ricin-filled pellet was fired into his leg using a modified umbrella. It has been estimated that as little as 500 micrograms of ricin could be fatal in humans if delivered by injection. Since ricin is easily extracted from the seeds of castor oil plants which are widely grown as an ornamental species and commercial crop, it has obvious potential for bioterrorist attacks. Until now, there has been no antidote to ricin poisoning, but French researchers have now described the first small molecules that are able to protect mice against its lethal effects.

Ricin and bacterial Shiga-like toxins exert their lethal effects by blocking protein synthesis. The proteins comprise two subunits, the A chain and the B-chain, which are linked by a disulphide bond. The B-chain facilitates cell entry and intracellular transport, and is reductively cleaved to free the A-chain which inactivates ribosomes and shuts down protein synthesis. To reach their cytosolic target, ribosomal RNA, the toxins follow the retrograde transport route from the plasma membrane to the endoplasmic reticulum, via endosomes and the Golgi apparatus.

Using a cell-based screen of over 16,000 compounds, the French team found two compounds that were able to block the transport of the toxins within the cell whilst showing very little cytotoxicity. The two compounds, Retro-1 and Retro-2, were shown to selectively block the intracellular transport of the toxins between early endosomes and the Golgi apparatus. Unlike other compounds that are known to block retrograde transport, Retro-1 and Retro-2 do not affect other intracellular trafficking and do not show any toxicity. Retro-2 was found to fully protect mice against a lethal dose of ricin, but only if administered before the ricin. Since the compounds act on host cell pathways rather than on the toxin itself, they should also defend against the Shiga-like toxins produced by pathogens such as E. coli, Shigella and Cholera.

The study is published in the journal Cell.

Stopping Cancer Spread

Fascin crystal structure — Human fascin complexed with macroketone, PDB ID=3LNA

The deadliest feature of cancer is its ability to spread, or metastasise. Migrastatin, a compound isolated from Streptomyces, was found to weakly inhibit tumour cell migration and, in 2005, researchers from Weill Medical College of Cornell University and the Sloan-Kettering Institute for Cancer Research described simplified analogues of migrastatin, including a compound they called macroketone, that inhibit mammary tumour metastasis in mice. Although the compounds were effective in preventing the spread of cancer cells, it wasn’t known how they achieved this. In a new study, published in the journal Nature, the team have revealed that macroketone exerts its anti-metastatic effect by targeting the actin-bundling protein, fascin. Cancer cells use invasive finger-like protrusions called invadopodia to spread into and degrade extracellular matrix and recent studies have shown that fascin is important for their assembly and stability.

Mice implanted with cancer cells and treated with macroketone lived out a full lifespan without any spread of the cancer whilst untreated animals all died from metastases. When treatment was delayed for one week after introduction of the cancer cells, metastasis was still blocked by more than 80%. Macroketone did not prevent implanted cancer cells from forming tumours or growing, suggesting that such compounds would need to be used in combination with chemotherapy drugs acting on the primary tumour. Because fascin is overexpressed in metastatic tumour cells but is only expressed at very low levels in normal epithelial cells, treatments that target fascin should have comparatively little effect on normal cells and may have fewer side effects than other treatments.

X-ray studies showed that macroketone binds to one of the actin-binding sites on fascin which prevents the actin fibres from bundling together and could form the basis for further drug design.

Role for TRPV3 in Skin and Hair Renewal

Transient Receptor Potential (TRP) ion channels are a large superfamily of transmembrane proteins which, amongst other functions, help an organism to sense its environment. The TRPV1 channel –which is activated by heat and also by capsaicin, the pungent component of hot chilli peppers – has been linked to the development of epithelial cancers.

A new study by researchers at the University of Michigan and Children’s Hospital Boston shows that the TRPV3 receptor – a molecular sensor for warm temperatures – plays a key role in maintenance of the skin barrier and hair growth and could perhaps also be linked to the development of skin cancer. Epidermal growth factor receptor (EGFR) and one of its ligands, transforming growth factor-α (TGF-α), were known to regulate skin and hair growth and the new study shows that TRPV3 is a key component of the EGFR signalling pathway. Activation of TRPV3 was found to lead to release of TGF-α, which activates EGFR. Activation of EGFR, in turn, increases TRPV3 channel activity, forming a positive feedback loop. Like animals with naturally occurring loss-of-function mutations in the genes for EGFR or TGF-α, TRPV3 knockout mice were found to have curly coats and whiskers.

In the TRPV3 knockout mice, the outer layer of the skin was found to be thinner than normal and to have a dry scaly texture which resulted from impaired terminal differentiation of keratinocytes. The study suggests that small molecule activators of TRPV-3 could be used to treat a variety of skin injuries and diseases such as burns, bed sores, eczema, psoriasis, itch, and fungal infections.

One cautionary note is that mice with overactive EGFR or TGF-α are hairless and develop skin cancers. Upregulation of EGFR has also been linked to a number of human cancers and drugs targeting EGFR are an expanding class of cancer treatments. Although mice overexpressing TRPV3 have not been shown to have an increased number of tumours, this is something that would need to be investigated further. The study is published in the journal Cell.

High Altitude Metabolism

The cellular responses to low oxygen levels (hypoxia), as occurs at high altitude, are critical for survival. The transcription factor, hypoxia inducible factor 1 (HIF-1), is a key player in this setting, upregulating genes that preserve function. Included in these are glycolysis enzymes, which allow ATP synthesis in an O₂-independent manner, and vascular endothelial growth factor (VEGF), which promotes angiogenesis. HIFs are also important in development and deletion of HIF-1 in mammals is perinatally lethal.

HIF-1 occurs as a heterodimer of HIF-1α and the constitutively expressed HIF-1β. Under normal oxygen conditions, HIF-1α is a substrate for HIF-1 prolyl hydroxylases and the asparagine hydroxylase, factor inhibiting HIF-1α (FIH). The action of the prolyl hydroxylases results in the targeting of HIF-1α by an E3 ubiquitin ligase and subsequent degradation by the proteasome, whilst hydroxylation by FIH represses activity of its carboxy terminal transactivation domain (CAD). Both hydroxylation processes therefore serve to down-regulate the activity of HIF-1. When oxygen levels are low, however, the prolyl hydroxylases and FIH become inactive since they are dependent on O₂.

A team led by researchers at University of California at San Diego have now reported on a FIH-knockout mouse. Despite the importance of HIF-1 in development, the FIH-deleted mice were healthy, although smaller than wild-type littermates. Where they differed significantly was in their metabolic profile. The FIH-null mice exhibited elevated metabolic rate, enhanced insulin sensitivity, hyperventilation and improved lipid and glucose homeostasis. On a high-fat diet, the animals were resistant to weight gain and had reduced central adiposity.

The team went on to explore the effects of tissue-specific FIH deletion, demonstrating that most of the features of the metabolic phenotype of the FIH-null mice could be replicated when only neuronal FIH was deleted.

The study, published in Cell Metabolism, identifies FIH as an essential regulator of metabolism and opens up the possibility of FIH inhibitors for the treatment of metabolic disorders.

Open Access Drug Target Database Launched

SciClips has launched an online database of drug targets, based on information collected from US and International patent applications. Currently the database contains around 4000 targets linked to their respective diseases and the data are updated on a weekly basis. As well as the association of the biological targets with diseases, the database is also organised according to type of drug (such as antibodies, proteins, siRNA, miRNA or small molecules).

All targets are linked to PubMed, Google Scholar, GeneBank, UniProt, the USPTO database, WO(PCT) database and Google Patents, allowing the user to access additional information, and searches can be conducted on the biological target, drug type or disease.

Whilst the web interface seems a little idiosyncratic, feedback from users will allow SciClips to refine it.

According to the SciClips website:

“SciClips is a web-based scientific platform for open innovation as well as for sharing information and ideas related to various scientific areas. Currently we are offering services and inviting ideas in several areas involving stem cells, proteomics, biomarkers, metabolomics and drug discovery tools. …. We believe that by offering scientific services to cutting edge research areas, we will be able to attract scientists (from) all over the world to think collectively, share ideas and achieve scientific breakthroughs. We will add services in other scientific areas in (the) coming months.”

Web-based Application for Tracking Infectious Diseases

The near pandemic of SARS (Severe Acute Respiratory Syndrome) that occurred in 2002/2003 underlined the need for vigilance in understanding and containing the global spread of pathogens. Genomic sequencing is an important tool in characterising novel pathogens but, to best understand the etiology of epidemics, genetic information must be integrated with host biology and geography. To this end, researchers at Ohio State University have developed a web-based application that allows users to visualise genetic and evolutionary information about organisms that cause infectious diseases as they spread across the globe.

The application, Supramap, was initially created to track the avian influenza virus, H5N1, and has more recently been use to monitor the spread of the H1N1 virus. With the H5N1 virus, mapping a mutation (Glu627Lys) in the PB2 protein supported the hypothesis that this mutation allows for increased replication of the virus in mammals but not that viruses with this mutation were moving westwards.

The application is able to calculate and project evolutionary trees for pathogens onto geographic information systems such as Google Earth. The resultant ‘weather maps for disease’ will allow scientists and public health officials to see when and where pathogens spread, jump between host species, and evolve to become drug-resistant. The development team hope to receive genomic and geographical data about pathogens from around the world and provide regularly updated maps that could determine global hotspots for the emergence of dangerous pathogens and indicate, for example, where and when particular antiviral drugs would be effective. The application is easy to use and researchers can submit raw sequence data to create a phylogenic tree that can be projected onto a map of the globe and viewed with Google Earth. As with all models, the more data that can be collected, the more sophisticated and accurate the analysis will be.

More details about the Supramap project are published in the journal Cladistics.

Lipid Genes Linked to Cancer

Although clinical and epidemiological studies have linked cancer with other chronic conditions such as inflammatory and metabolic diseases, the pathways linking different diseases are poorly understood. Inflammation is commonly associated with the development and progression of cancer and increased cancer risk is also linked to metabolic syndrome which encompasses obesity, type II diabetes, high cholesterol, and atherosclerosis. To overcome limitations in previous approaches to identifying common genes and signalling pathways, researchers at Harvard Medical School have carried out transcriptional profiling in two experimental isogenic models of cellular transformation. Using two isogenic models in which the transformed and non-transformed tissues are genetically identical makes it is easier to identify genes involved in transformation.

The team used a computational approach to organise the genes identified by transcriptional profiling into networks with central nodes. Comparison with a gene set describing metabolic syndrome revealed a high overlap between the central nodes of cancer and metabolic syndrome. Inflammatory factors such as IFN-γ, IL-1β, IL-6, and NF-κB as well as insulin and low-density lipoprotein (LDL) appeared as central nodes in cancer gene networks, suggesting the importance of inflammatory processes in both cancer and metabolic diseases and also a link between protein and lipid metabolism and cellular transformation. Lipid-related genes that had not previously been linked to cancer included OLR1, SNAP23, VAMP4, SCD1, SREBP1 and GALNT2.

The similarities between the pathways in cancer and metabolic diseases led the team to test whether drugs used to treat inflammation or aspects of metabolic disease might also affect cellular transformation and tumorigenicity. Four of the compounds that performed best in the cell experiments, metformin, sulindac, simvastatin, and cerulenin were tested for their ability to suppress tumour growth in nude mice: tumour growth was completely suppressed by metformin and sulindac and significantly delayed by cerulenin and simvastatin, suggesting that drugs designed to combat metabolic diseases may also be useful in treating some types of cancer.

The study is published in Cancer Cell.