A year ago, when researchers at Purdue University argued that environmental standardisation in laboratory experiments involving mice was likely to lead to more, rather than less, variation between different laboratories, they met with some resistance since it was not clear what factors should be varied to improve reproducibility. Following an analysis of data from behavioural tests commonly used in drug discovery studies, they have now shown that introducing only two controlled environmental variables to preclinical studies using mice can greatly reduce false positives and the number of animals needed for testing. The tests, which compared behaviours between two inbred strains of mice, were repeated in four different model laboratories that varied in details such as background noise, the age of the mice, environmental enrichment, familiarity with handler, lighting levels and cage size. In each laboratory, one group of mice (standardised) were treated identically whilst the other group (heterogenised) were tested under four different sets of conditions produced by varying two environmental factors in a controlled manner. Mice of the same strain would have been expected to show the same behaviours in each laboratory but, in 33 out of 36 behavioural characteristics such as fear and curiosity, the standardised group showed as much as five times more variation between laboratories compared with the heterogenised group.

When conditions are highly standardised, the variation in data produced within a particular laboratory will be very low, but variations between laboratories will be large and unpredictable. The researchers believe that tests in mice using a heterogeneous test design more closely resemble human clinical trials and should reduce both the number of animals needed for preclinical studies and the number of false positives. A reduction in false positives could have important implications for reducing the number of compounds that fail in expensive downstream clinical trials.

The study is published in Nature Methods.

In back-to-back papers published in the Proceedings of the National Academy of Sciences, researchers describe retro-inverted peptide mimetics targeting the vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) pathways. The team begin by screening a phage display library to identify peptides that bind to the target receptor and refining the hits by structural and functional analysis. Since natural peptides are often unstable to proteolysis, the amino acid retro-inversion method (ie substitution of D-amino acids for L-amino acids in conjunction with reversal of chain direction) was used to produce molecules with greater resistance to proteolysis. The side chain topology of the resulting peptide mimetics is similar to that of the original peptide – albeit with the amide bonds and terminal charges reversed.

The formation of new blood vessels has been associated with a number of pathologies including cancer and diseases of the eye such as diabetic retinopathy and age-related macular degeneration. VEGF comprises a family of five growth factors that promote angiogenesis by binding to, and selectively activating, several membrane-bound tyrosine kinase receptors (VEGFR-1, -2, and -3) and neurophilins (NRP-1 and -2). The minimal structural requirement for binding of a small peptide fragment to VEGFR-1 and NRP-1 was determined to be the tripeptide, RPL. The retro-inverted tripeptidomimetic, D-LPR was found to be resistant to proteolysis and to bind effectively to VEGFR-1 and NRP-1. In competition binding experiments, the IC₅₀ values of RPL for VEGFR-1 and NRP-1 were 30 nM and 4 pM and the IC₅₀ values of D-LPR for VEGFR-1 and NRP-1 were 2 pM and 2 pM. D-LPR was shown to be effective in three animal models of angiogenesis, including a mouse model of retinopathy. When administered topically, D-LPR led to a significant (ca 50%) reduction in angiogenesis, suggesting that the peptide mimetic can penetrate the vitreous humour and may provide a lead for the development of soluble and permeable small drug molecules that can be administered in eye drops.

EGFR, a tyrosine kinase receptor, is abnormally activated in many types of epithelial tumours, including including lung, colon, and head and neck cancers. Although, theoretically, the EGFR pathway could be blocked by three drug classes – antibodies, kinase inhibitors or soluble ligand receptor traps/decoys – so far no EGFR decoy has been developed. In the second paper, the team describe small molecules that mimick EGFR and behave as soluble decoys for EGFR ligands, EGF and TGFα. The cysteine-bridged pentapeptide, CVRAC, was found to bind specifically to EGFR ligands and both CVRAC and the retro-inverted derivative, D-CARVC, markedly reduced proliferation of tumour cell lines. In immunocompetent female mice bearing mammary tumours, mice treated with D-CARVC had significantly smaller tumour volumes than control mice.

Onchoceriasis – also known as river blindness – is the world’s second leading infectious cause of blindness. The disease is caused by the nematode, Onchocerca volvulus, and is transmitted to humans through the bite of a blackfly. Once inside the body, the female worm produces thousands of larval worms (microfilariae) which migrate to the skin and eyes. When the microfilariae die, they cause intense itching and a strong immune response that can destroy nearby tissue, leading eventually to blindness and disfiguring skin lesions. Control programmes have involved the use of larvicides to reduce blackfly populations and the use of ivermectin to treat infected people and limit the spread of disease. Ivermectin is most effective against the larval stage of the worm and is believed to kill the parasites by activating glutamate-gated chloride channels which are specific to invertebrates.

A team led by researchers at the Scripps Institute has now focused on a new way to kill the parasite. The protective outer cuticle of the worms is made of chitin and two classes of enzymes – chitin synthases and chitinases – are known to be critical for chitin formation and remodelling. One chitinase, OvCHT1, is expressed only in the infective third-stage larvae and is believed to be involved in development and host transmission. The team screened a small library of compounds for activity against OvCHT1 and found that closantel was able to inhibit the enzyme. When closantel was tested on cultured third-stage larvae, the compound prevented the larvae from moulting and developing into adult worms. Since the mechanism of action of closantel is completely different to that of ivermectin, it – or other chitinase inhibitors – could potentially be used to treat ivermectin-resistant worms. Closantel is a broad-spectrum anti-parasitic agent currently used in some countries in veterinary medicine.

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

In 2008, researchers led by a team at Columbia University showed that, by turning on or off production of serotonin in the gut, they could control bone formation. Serotonin signals to cells in the skeleton to slow production of new bone and, by turning off the intestine’s release of serotonin, the team was able to prevent osteoporosis in mice undergoing menopause. The team have now shown that daily oral administration of LP-533401 for 6 weeks is effective both prophylactically and therapeutically against osteoporosis in ovariectomized mice.

LP-533401 inhibits tryptophan hydroxylase-1 (TPH-1), the first enzyme in gut-derived serotonin biosynthesis. TPH-1 is mostly expressed in peripheral tissues such as the gut, whilst TPH-2 is the major isoform in the central nervous system. Although LP-533401 inhibits human TPH-1 and TPH-2 with similar potency (K_i ~ 0.7µM) in vitro, it selectively lowers serotonin levels in the gut whilst leaving levels in the brain unchanged, likely because the compound does not cross the blood-brain barrier. LP-533401 and an ethyl ester pro-drug were originally developed to treat gastrointestinal diseases such as irritable bowel syndrome and to reduce chemotherapy-induced vomiting and nausea.

Although much work will need to be done before trials can be carried out in patients, the present study, which is published in Nature Medicine, demonstrates that pharmacological inhibition of synthesis of gut-derived serotonin could become a new anabolic treatment for osteoporosis. Most osteoporosis drugs only prevent the breakdown of old bone and are not able to stimulate the growth of new bone.

Although a variety of broad-spectrum antibiotics have been developed, broad-spectrum antiviral agents have proved more difficult to identify. Effective treatments have been developed for individual viruses such as HIV, herpes viruses and influenza viruses – and vaccines have also been developed against papilloma viruses and herpes viruses – but there is a need for small molecules that are able to treat a range of viral infections and could also be used against newly emerging viruses.

Researchers led by a team at UCLA have now identified a compound, LJ-001, that can treat a range of enveloped viruses. The team screened a library of around 30,000 compounds against Nipah virus, a pathogen that was first identified in 1998 and causes severe disease in both animals and humans. Further tests showed that LJ-001 was also effective against other enveloped viruses including Ebola virus, HIV, hepatitis C virus, West Nile virus, Rift Valley fever virus, yellow fever virus and influenza A virus, but had no effect against non-enveloped viruses. The compound interacts with the viral lipid envelope and inhibits viral entry at a step after virus binding but before virus–cell fusion.

Although LJ-001 also binds to cellular membranes, the team believe that its low toxicity can be attributed to the fact that metabolically active cells are able to repair their membranes whilst static viruses are not. LJ-001showed no overt toxicity at effective anti-viral concentrations in either in vitro or in vivo studies, and pretreatment of mice with LJ-001 prevented virus-induced mortality from Ebola and Rift Valley fever viruses.

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

Scientists at Vanderbilt University have previously used zebrafish embryos to identify compounds that interfere with signalling pathways involved in early development – pathways that also play a role in many disease processes. One of these compounds, dorsomorphin, was shown to block bone morphogenetic protein (BMP) signalling, a pathway that is involved in bone and cartilage formation and that has also been linked to anaemia and inflammatory responses. Subsequent studies showed that dorsomorphin also blocked the vascular endothelial growth factor (VEGF) type-2 receptor and disrupted angiogenesis.

To identify more selective compounds, the team turned again to zebrafish embryos. It was quickly discovered that the two effects could be separated, with some compounds only affecting patterning and some only affecting angiogenesis. The former were shown to be potent and selective inhibitors of BMP signalling and the latter to be selective VEGF inhibitors. As well as identifying a VEGF inhibitor that outperformed a compound that had entered phase III clinical trials, the team also discovered a BMP inhibitor, DMH1, which exclusively targets the BMP pathway. Using zebrafish embryos for structure-activity analyses allows selectivity and bioavailability to be assessed at the same time as efficacy and the team believe that zebrafish provide an attractive complementary platform for drug discovery. The potential of small molecule signalling inhibitors is often limited by off-target activities and zebrafish provide a very good model for assessing selectivity since compounds that hit multiple targets are toxic to the embryos.

The study is published in ACS Chemical Biology.

On Monday the European Molecular Biology Laboratory’s European Bioinformatics Institute (EMBL-EBI) announced ChEMBLdb, a drug discovery database containing information on over half a million compounds. The database was transferred to EMBL in July 2008 from biotech company, Galapagos, via a £4.7 million strategic award from the Wellcome Trust. Since acquisition, the ChEMBL team have added data on an additional 100,000 compounds prior to the launch.

The database is composed of drugs and small-molecule drug-like compounds together with information on their biological targets, effects on cells and whole organisms as well as their absorption, distribution, metabolism, excretion and toxicity (ADMET) properties. The data are abstracted and curated from the primary literature.

Dr John Overington, leader of the ChEMBL team at EMBL-EBI, said:

“We hope ChEMBLdb will assist the translation of genomic-based insights into innovative drug therapies. We are pleased that there has already been big demand for ChEMBLdb data – not only from large pharmaceutical companies but also from academic institutions and small companies who will particularly benefit from free access to the data.”

The database, accessible at www.ebi.ac.uk, can be searched for biological targets by keyword or BLAST; compound searching can be by keyword, structure or substructure. In addition, the launch of ChEMBLdb is accompanied by the release of Kinase SARfari, an integrated resource of sequence, compound and screening data from a variety of sources for the protein kinases, a key family for drug discovery.

The cyclic nucleotide phosphodiesterases (PDEs) are important regulators of signal transduction and selective inhibitors of the different subtypes have great clinical potential. PDE4 inhibitors are expected to be beneficial in the treatment of inflammatory and respiratory diseases such as asthma and COPD as well as CNS disorders including schizophrenia, depression, and Alzheimer’s disease but their potential has so far been limited by the incidence of side effects, particularly emesis. The emetic response is mediated in part by a brainstem noradrenergic pathway and, for non-CNS indications, can be reduced by limiting distribution of inhibitors to the brain. Active site directed PDE4 inhibitors completely inhibit enzyme activity at high concentrations but researchers at Emerald Biostructures (formerly deCODE biostructures) have now identified allosteric small molecule modulators of PDE4 with reduced potential for side effects. The four PDE4 variants (PDE4A, B, C, and D) all contain signature regulatory domains called upstream conserved regions 1 and 2 (UCR1 and UCR2). UCR2 is needed for high-affinity binding of the PDE4 inhibitor rolipram and X-ray crystallographic structures revealed that small molecule inhibitors bind to UCR2, thereby controlling access to the active site. The team used the structural data together with supporting mutational data to design PDE4 allosteric modulators that only partially inhibit cAMP hydrolysis. The modulators were shown to be potent in cellular assays as well as in vivo cognition tests and to have greatly reduced potential for emesis in several species. The authors hope that their work will lead to the identification of PDE4 modulators with reduced potential for emesis that can be used to treat disorders where brain distribution is needed. The study is published in the December 27^th advance online issue of Nature Biotechnology.

Cystic fibrosis is an inherited disease that affects about 70,000 children and adults worldwide. The condition is caused by a mutation in the gene cystic fibrosis transmembrane conductance regulator (CFTR). Defects in the protein product – which transports chloride ions – lead to unusually thick, sticky mucus that clogs the lungs and also blocks the ducts of the pancreas, preventing digestive enzymes from reaching the intestine. The most common mutation, which causes a severe form of the disease, is a deletion of a phenylalanine residue at position 508 of the protein (DF508 CFTR) which results in the absence of CFTR protein at the cell surface. Current treatments for cystic fibrosis focus primarily on managing the symptoms and drugs that are able to restore function of DF508 CFTR protein at the cell surface, which would benefit the majority of cystic fibrosis patients, are not available.

A new study by Scripps scientists working in collaboration with investigators from the US and Canada has now shown, however, that the HDAC inhibitor, suberoylanilide hydroxamic acid (SAHA), can restore about 28 percent of normal ion channel function to cultures of lung epithelial cells from patients with the DF508 CFTR mutation. The team speculated that mutant CFTR proteins – which could still provide some useful function – were being degraded by the endoplasmic reticulum and reasoned that modifying HDAC function might rebalance proteostasis networks in the cell to favour functional restoration. When the cells were treated with SAHA, DF508 CFTR was expressed at the cell surface at comparable levels to wild-type protein. Inhibition of HDAC7 appeared to be largely responsible for this effect although little is known about the physiological role of HDAC7. Since it is known that cystic fibrosis patients with 15-30% of normal CFTR function have milder disease, the level of functional restoration provided HDAC7 inhibition has the potential to provide significant benefit.

SAHA (vorinostat) is currently approved for the treatment of cutaneous T-cell lymphoma (CTCL), but the researchers caution that much more work will be needed before this approach can lead to new therapies for cystic fibrosis.

The study, published in Nature Chemical Biology, takes a new approach to drug discovery by targeting cellular proteostasis and could have application in numerous chronic diseases that are characterised by protein misfolding.

Around 85% of non-Hodgkin’s lymphomas in the United States are B-cell lymphomas and, of these, diffuse large B-cell lymphoma (DLBCL) account for about one in three cases. DLBCL is a fast growing lymphoma and only about half of people with this type of lymphoma are cured by current treatments, which include radiation therapy, chemotherapy and monoclonal antibodies.

Researchers, including scientists from Weill Cornell Medical College, have now discovered that the heat shock protein inhibitor, PU-H71, selectively kills DLBCLs that depend on the B-cell lymphoma 6 protein (BCL-6) transcriptional repressor. BCL-6 is involved in the pathogenesis of around 70% of cases of DLBCL. BCL-6 and heat shock protein (Hsp90) were almost invariantly co-expressed in the nuclei of primary DLBCL cells and a complex of Hsp90 and BCL-6 was found to stabilise BCL-6 mRNA and protein. Hsp90 inhibitors allowed transcription of genes normally repressed by BCL-6 and a stable mutant of BCL-6 was found to rescue DLBCL cells from Hsp90 inhibitor–induced apoptosis. In mouse xenograft models, PU-H71 was shown to preferentially accumulate in lymphomas compared to normal tissues, and led to almost complete tumour regression by allowing reactivation of key BCL-6 target genes and inducing apoptosis. PU-H71 shows very low toxicity in animal models and the researchers hope that the compound will be similarly well tolerated and efficacious in human patients.

The study is published in Nature Medicine.

PU-H71 has previously been shown to induce complete responses in triple-negative breast cancer models. Triple-negative breast cancers are defined by a lack of expression of estrogen, progesterone or HER2 receptors and are currently treated with conventional chemotherapy which is effective only in some patients, leaving others with high rates of early relapse.