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.
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 27th 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.
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.
Transcription factors – proteins that bind to specific DNA sequences and promote or block transcription – are essential for correct regulation of gene expression. Since many transcription factors are tumour suppressors or oncogenes, and mutations are associated with the development of different types of cancer, they have been the subject of intense interest. As a class, however, they have proved to be difficult drug targets since the vast majority of transcription factors lack an appropriate binding pocket for targeting by small molecules and, since most are intracellular, they are not suitable for antibody-based therapies. A team of US scientists have now discovered a way to modulate activity of the transcription factor, Notch, which is often mutated or damaged in patients with T-cell acute lymphoblastic leukaemia (T-ALL). Abnormalities in Notch signalling also underlie a variety of other cancers, including lung, ovarian, pancreatic and gastrointestinal cancers. The structure of Notch binding to other proteins in the transactivation complex revealed a long shallow helical groove at one of the protein-protein interfaces, and the team designed compounds that bind in this region and prevent complex assembly. The compounds are ‘stapled peptides’, held in a complementary helical shape by hydrocarbon crosslinks. Experiments in cultured T-ALL cells, and in a mouse leukaemia model, showed that one of the compounds, SAHM1, was able to limit the growth of cancer cells controlled exclusively by Notch, but did not inhibit the growth of cells that were not regulated by Notch. At a molecular level, the peptide reduced expression of genes that are controlled directly and indirectly by Notch.
The direct inhibition of Notch did not produce the gastrointestinal toxicity which has been observed with inhibition of Notch processing by γ-secretase inhibitors, but further experiments will be needed to ensure that side effects are not caused by blocking this highly conserved and important signalling pathway.
As well as identifying a compound that inhibits Notch signalling, the work could also lead to inhibitors of other transcription factors that assemble in a similar way to Notch and are associated with conditions such as diabetes and autoimmune diseases. Despite being considerably larger than typical small molecule drugs, the ‘stapled peptides’ are able to penetrate cells since they are taken up by an active transport mechanism.
Spinal Muscular Atrophy (SMA) describes a group of diseases where motor neurons of the spinal cord and brain stem, which are critical for stimulation of muscle cells, degenerate and die. Lacking the appropriate input, the muscle cells become much smaller (atrophy) and patients display symptoms of muscle weakness. Affected muscles are those involved in voluntary movement and patients may have difficulty swallowing, breathing, crawling, walking and with head/neck movement. SMA is an autosomal recessive genetic disease and for a child to be affected both parents must be carriers of the abnormal gene and both must pass this gene on to their child. The incidence of SMA is estimated at 1 in 6000 births and this condition is responsible for the death of more infants than any other genetic disease.
SMA results when the SMN1 (survival of motor neuron 1) gene, which encodes survival of motor neuron (SMN) protein, is missing or mutated. SMN is critical to the survival and health of motor neurons. The closely related survival of motor neuron SMN2 gene is retained in all SMA patients but does not produce sufficient SMN protein to prevent the development of clinical symptoms. Although SMN2 differs from SMN1 by only a single nucleotide, the change affects the efficiency with which exon 7 is incorporated into the mRNA transcript. As a result, SMN2 produces less full-length mRNA and protein than SMN1.
In 2001, researchers at Ohio State University showed that aclarubicin was able to restore levels of SMN in a mouse model by altering the incorporation of exon 7 into SMN2 transcripts. Although aclarubicin is too toxic to consider for development, the work prompted scientists at Paratek Pharmaceuticals to screen related tetracycline analogues. This has now resulted in the identification of PTK-SMA1, a synthetic tetracycline-like compound, as a lead candidate. PTK-SMA1, like aclarubicin, increases levels of SMN by correcting SMN2 splicing. The study, conducted in collaboration with scientists at Cold Spring Harbor and Rosalind Franklin Univeristy, is published in Science Translational Medicine.
Further collaborative research to progress the program to IND filing is being supported by a five-year, multi-million dollar cooperative agreement from the National Institute of Neurological Disorders and Stroke (NINDS) and by the Families of SMA funding program.
The renin-angiotensin system was originally believed to be a relatively straightforward cascade involved primarily in controlling blood pressure and was exploited to provide a number of antihypertensive medicines. Angiotensinogen is cleaved by renin to give angiotensin I (Ang I), followed by further cleavage of Ang I by angiotensin-converting enzyme 1 (ACE) to give the octapeptide angiotensin II (Ang II) which binds to angiotensin receptor 1 (AT1R) or 2 (AT2R). AT1R mediates most of the cardiovascular effects of Ang I and activation leads to increased blood pressure which can be countered by the action of renin inhibitors, ACE inhibitors or AT1R antagonists.
More recently, a parallel pathway in which Ang I is cleaved by angiotensin-converting enzyme 2 (ACE2) to give the heptapeptide, angiotensin 1-7 [Ang-(1-7)], has been discovered. This second pathway, in which Ang-(1-7) binds to the Mas receptor, is believed to act as a counter-regulatory axis to the ACE 1 – Ang II – AT1R axis. Roles have also been proposed for Ang-(1-7) and Mas in kidney disease, liver disease, and spermatogenesis.
Ang-(1-7) is also known to have anti-proliferative properties and to inhibit angiogenesis and researchers at Wake Forest University School of Medicine have now shown that Ang-(1-7) reduces lung tumour growth and inhibits blood vessel formation in mice. The team had previously shown that Ang-(1-7) inhibits the growth of human lung cancer cells in vitro and reduces the size of human lung tumour xenografts in vivo. In the present study, daily subcutaneous administration of Ang-(1-7) for six weeks was found to significantly reduce tumour growth by human lung carcinoma A549 cells and also markedly decrease vessel density compared with saline treated controls. The study, which is published in the August issue of Molecular Cancer Therapeutics, suggests that Ang-(1-7) may provide a novel and effective treatment for lung cancer. The team have also shown that Ang-(1-7) is effective against breast, colon and brain tumours. A clinical trial of Ang-(1-7) has been completed at the School of Medicine and the results are currently being reviewed.
This is not a question that the FDA normally asks before approving a new drug, but researchers at Stanford University Medical School have called for a change in labelling to include information about how effective the new drug is compared with existing treatments. And, if this information is not available, the researchers believe that the label should clearly say so. With the exception of situations where it would be unethical to withhold active treatment from one study group, as in the case of cancer patients or those with HIV/AIDS, the FDA requires only that a drug should be deemed safe and effective in order to receive marketing approval. In many cases, ‘efficacy’ is determined solely on the basis of superiority to placebo.
Writing in the New England Journal of Medicine, the authors suggest that including comparative efficacy information on labels would allow more informed choices by clinicians, patients and providers and also curb increases in healthcare costs. The authors cite several reasons for the current practice of carrying out placebo-controlled trials; smaller sample size, reduced cost, and lowered risk of generating unexpected unfavourable results (for example, neither active comparator found to be better than placebo). Drug development is an inherently long and risky business and, although some products represent true therapeutic breakthroughs, many offer little benefit over existing therapies. The authors argue that the current regulatory climate provides few incentives to conduct active-comparator trials and favours creation of products that differ minimally from existing therapies.
Whitehead Institute researchers have described a new drug discovery technique which uses yeast cells to both synthesise and screen novel compounds. Writing in the journal Nature Chemical Biology, the team have demonstrated that they can negate the toxic effects of α-synuclein in a yeast model that mimics much of the cellular pathology of Parkinson’s disease. α-Synuclein accumulates in vulnerable brain cells in patients with Parkinson’s disease, and the team had previously shown that yeast cells engineered to express large amounts of α-synuclein do not survive. In the new study, yeast cells were engineered to produce cyclic peptides – which target protein-protein interactions – and the α-synuclein was then switched on. The cells that produced cyclic peptides that protect against α-synuclein toxicity survived: the rest died. Out of a library of millions of cyclic peptides, only two were found to rescue yeast cells from α-synuclein toxicity. Although it is not yet clear how the cyclic peptides protect the cells – they were shown not to affect vesicle trafficking – both peptides share a structural motif with thioredoxins, proteins that act as antioxidants; metal transport proteins and proteins that regulate gene activity. The team are now working to determine the precise mechanism of action and to develop new analogues of the peptides.
In a follow-on study carried out by researchers at the University of Alabama, these two cyclic peptides were also found to protect dopaminergic neurones in a C. elegans model of Parkinson’s disease.
The new technique is rapid and inexpensive compared with other methods of lead discovery, and should be applicable to other diseases where key aspects of the pathology can be modelled in yeast or mammalian cells.