News – Page 11 – Drug Discovery Opinion

Chymase Inhibitors Could Improve Outcomes for Heart Failure Patients

Frozen Heart — Image: Flickr - laradanielle

Angiotensin converting enzyme (ACE) inhibitors are widely used for the treatment of hypertension and heart failure. The beneficial effects of ACE inhibitors in heart failure are believed to be linked to reduced local production of the potent vasopressor, angiotensin II (AII), but the realisation that another enzyme, chymase, can also produce AII suggests that chronic ACE inhibitor treatment may not completely suppress production of AII.

Chymase is a serine protease found mainly in mast cells but also in the cardiac interstitial space and in some cardiac endothelial cells. Scientists at Emory University, the University of Alabama, Birmingham, and Fukuoka University have now shown that adding a chymase inhibitor to ACE inhibitor treatment can significantly improve recovery of heart function in animals following a heart attack.

The team showed that chronic ACE inhibition produced a bradykinin-dependent release of a chymase (mouse mast cell protease-4, mMCP4) from mast cells in conscious mice, which maintains levels of AII in the interstitial fluid. ACE inhibition also decreases bradykinin degradation and the release of chymase into the left ventricular interstitial fluid may be the result of activation of bradykinin B2 receptors on mast cells. In hamsters, chronic ACE inhibition was found to improve left ventricular function and reduce the amount of dead tissue and scarring after myocardial infarction (MI) and the beneficial effects of chymase inhibition were greater when combined with ACE inhibition. Chymase inhibition may also affect maturation of proteases involved in tissue remodelling, independently of increases in AII levels.

Although recognising that clinical validation is still needed – and that there are no chymase inhibitors available for use in the clinic – the authors propose that addition of a chymase inhibitor to ACE inhibitor therapy may improve outcomes for patients with cardiac diseases including hypertensive heart disease, viral myocarditis, and atherosclerotic coronary artery disease, the prelude to MI.

Sibling Rivalry in the Bacterial World

Competing *Paenibacillus dendritiformis* colonies

Credit: Eshel Ben-Jacob

Bacterial colonies cultured on agar avoid each other when forced to compete for nutrients, but the mechanism behind the observed growth inhibition has been unclear. Now a new study by collaborating scientists at UC San Diego, University of Texas and Tel Aviv University has explored the behaviour of Paenibacillus dendritiformis cultures, identifying the factors responsible.

The team found that it was not a shortage of food that halted the growth. They found nutrients in the no-man’s land between the colonies, but also a protein that wasn’t present elsewhere on the dish. When a sample of the purified protein was introduced to a fresh dish inoculated with P. dendritiformis, the bacteria formed a lopsided colony that shied away from the spot. In addition, the bacteria at the edge of the colony closest to the suspect protein were dead.

Analysis of the secretions from P. dendritiformis identified the protease, subtilisin, and a 12 kDa protein, termed sibling lethal factor (Slf). Whilst subtilisin promotes growth and expansion of P. dendritiformis colonies, Slf lyses the bacterial cells in culture. Slf is encoded by a gene belonging to a large family of bacterial genes of unknown function, and the gene is predicted to encode a protein of approximately 20 kDa. The team generated recombinant 20 kDa protein, which was found to be inactive. Exposure to subtilisin, however, resulted in cleavage to the active, 12 kDa form. The experimental results, combined with mathematical modelling, show that subtilisin regulates growth of the colony. Below a threshold concentration subtilisin promotes colony growth and expansion, but once it exceeds a threshold, as occurs at the interface between competing colonies, Slf is then secreted into the medium to rapidly reduce cell density by lysis of the bacterial cells. The presence of genes encoding homologs in other bacterial species suggests that this mechanism for self-regulation of colony growth might not be limited to P. dendritiformis.

The study is published in PNAS.

New, Non-Obvious, Models for Human Disease

Arabidopsis Thaliana — Mustard plant, *Arabidopsis thaliana*, could be used to model human disease

Image: Wikimedia – Alberto Salguero

Model organisms used in the study of human disease have, up until now, generally closely resembled the disease being studied but scientists in the US have described a new way of identifying other, less obvious, disease models. Although the functions of a particular protein are generally conserved at the molecular level, this is not necessarily true at the level of the organism and disruption of protein function can lead to radically different phenotypic outcomes in different species.

Orthologous phenotypes (phenologs) are defined as phenotypes related by the orthology of the associated genes in two organisms although the observed phenotypes may be very different, depending on the organism-specific role played by each set of genes. The team examined human disease-gene associations and gene-phenotype associations in mice, C. elegans and yeast, looking for overlaps. As well as correctly identifying many known mouse models of human disease, the analysis also identified completely new phenologs. Two processes that were found to share 5 genes were abnormal angiogenesis in mutant mice and reduced growth rate of yeast deletion strains in the presence of the cholesterol-lowering drug, lovastatin. The set of genes regulates cell wall stress and biogenesis in yeast cells and regulates proper formation and maintenance of blood vessels in mice, suggesting that budding yeast – whilst obviously lacking blood vessels – could potentially model mammalian angiogenesis. The model predicts that some of the other genes associated with lovastatin sensitivity in yeast should also be involved in angiogenesis, and the team were able to identify a previously unrecognised role for the gene SOX13 in angiogenesis.

To test whether other very distantly related species could serve as human disease models, the team looked for phenologs between the mustard plant, Arabidopsis thaliana, and fungi and animals. The human-plant phenologs suggested associations between specific plant mutational phenotypes and a variety of human diseases, including cancers, peroxisomal disorders and birth defects. One striking plant-human phenolog linked plant gravitational growth responses to Waardenburg syndrome.

In spite of the extremely different phenotypes, phenologs can identify functionally coherent gene sets that predate the divergence of plants and animals and lead to novel, non-obvious models of human disease.

The study is published in PNAS.

Proof of Concept for siRNA in Melanoma

Green and Red Lights — Image: Flickr - shikeroku

By far the majority of marketed drugs modulate the activity of proteins. Since the discovery of RNA interference (RNAi) over 10 years ago, a major goal has been to develop agents which will selectively block the synthesis of target proteins. Small interfering RNAs (siRNAs) occur naturally and are believed to have evolved as a defence mechanism against viral infection but synthetic siRNAs could potentially be used to silence any target gene. Although the idea is not conceptually difficult and many cancer-associated genes have been silenced in laboratory experiments, there have been a number of obstacles in developing RNAi for clinical use, not least of which is how to deliver the siRNAs to the target cells.

A team led by scientists and clinicians at the California Institute of Technology (Caltech) has now published the results of a small phase I clinical trial in patients with skin cancer showing that targeted nanoparticles can traffic into tumours and deliver siRNAs in a dose-dependent fashion when administered intravenously. The siRNA-containing nanoparticles used in the study are being developed by Calando Pharmaceuticals, Inc. and the technique used for detecting and imaging the nanoparticles inside cells from tumour biopsies was developed at Caltech. Although phase I studies are primarily designed as safety studies, the team was able to demonstrate that, in one patient who received the highest dose of nanoparticles, the target mRNA (M2 subunit of ribonucleotide reductase (RRM2)) had been cleaved at the predicted position and that protein levels had also been reduced.

Although more trials will be needed to show that such treatment is safe and effective, the study provides the first evidence that nanoparticles and RNAi can be combined to reduce expression of cancer-associated genes in human patients.

The results are published in the journal Nature.

Teenagers Really Are Different

Puberty is a time of great physical and emotional changes and studies have shown that some skills are most easily acquired before – or after – puberty. Working with mice, scientists at SUNY Downstate Medical Center in Brooklyn have now been able to suggest a reason for the observed reduction in learning ability seen in adolescents. They have shown that there is a temporary increase in levels of the α4βδ GABA-A receptor in the hippocampus during puberty. The hippocampus plays key roles in spatial learning and memory and increases in levels of the receptor reduce brain excitability and impair spatial learning. Levels of α4βδ GABA-A receptor were found to increase at puberty, falling back to an intermediate level when the animals reached maturity. Pubescent mice were found to be much less able to master a test of spatial learning than prepubescent animals. The team also showed that the learning disability could be reversed by administration of the stress steroid, THP. In human children and adults, THP reduces brain activity and has a tranquilizing effect; in pubescent mice, the hormone increases activity in the hippocampus and has the opposite effect.

The study suggests that intrinsic learning mechanisms alter during adolescence and that the temporary decline in learning ability might be reversed in middle school by different teaching and motivation strategies which involve mild stress. If findings from the mouse studies are applicable to human teenagers, compounds targeting the α4βδ GABA-A receptor may also prove useful, especially for adolescents with learning difficulties.

The study is published in the journal Science.

Novel Strategy to Fight TB

The bacterium responsible for tuberculosis (TB), mycobacterium tuberculosis (Mtb), is notoriously difficult to kill. The most commonly used antibiotics, rifampicin and isoniazid, need to be used for extended periods of time (typically 6-24 months) to effectively eliminate infection. In addition, emergence of antibiotic-resistant strains is an increasing problem.

Researchers at Albert Einstein College of Medicine of Yeshiva University have now identified a new biochemical pathway in Mtb and two novel ways to kill the bacterium. The pathway involves four enzymatic steps in the conversion of the disaccharide, trehalose, to α-glucan mediated by TreS, Pep2, GlgE (which has been identified as a maltosyltransferase that uses maltose 1-phosphate) and GlgB. Focusing on GlgE, the researchers found that blocking the enzyme induced toxic accumulation of maltose-1-phosphate, killing the bacteria in vitro and in a mouse model of infection. Inhibition of another enzyme in the pathway was non-lethal until combined with inactivation of Rv3032, a glucosyltransferase involved in a distinct α-glucan pathway. Inhibition of Rv3032 alone was also non-lethal to the bacteria.

The research validates inhibition of GlgE as therapy for TB but also highlights the potential for targeting two α-glucan pathways – a strategy that potentially leads to reduced incidence of resistance. Both approaches are also distinct from the mechanisms of currently used antibiotics.

The study is published in Nature Chemical Biology.

Repairing the Damage

mouse and salamander — Salamander photo: Flickr - brian.gratwicke; Mouse photo: Wikimedia - Ilmari Karonen

Although some species of amphibians – such as salamanders and newts – are able to regenerate lost or damaged tissue, the capacity for regeneration diminished as vertebrates evolved and adult mammals generally have limited regenerative capacity. Over a decade ago, it was discovered that the Murphy Roths Large (MRL) mouse strain was able to regenerate new epidermis, new hair follicles, and new cartilage to repair punch holes in the ears, unlike other strains of mice that close the holes with scar tissue. The MRL mice were later shown to have the ability to repair damaged heart muscle and spinal cord with restoration of normal structure and function and to have some limited capacity for digit re-growth. Scientists have been trying to identify the gene or genes that are responsible for the increased capacity for regeneration in MRL mice and researchers at the Wistar Institute and Washington University have now shown that the p21 gene is involved in regulating the regeneration process.

The team showed that p21, a cell cycle regulator, was found to be consistently inactive in cells from MRL mice. When they looked at p21 knockout mice, they found that, unlike normal mice which heal wounds by forming a scar, mice that lack p21 begin by forming a blastema, a mass of cells capable of rapid growth and de-differentiation which behave more like embryonic stem cells than adult mammalian cells. The p21 knockout mice were able to replace missing or damaged tissue with healthy tissue that showed no signs of scarring.

Since the cyclin-dependent kinase p21 is one of the best characterized downstream targets of the tumour suppressor p53, knockout of p21 might be expected to increase the incidence of cancer and other studies have suggested that p21-deficient mice develop tumours at an earlier age than their wild-type counterparts and are more susceptible to the effects of some carcinogens. Although increased DNA damage was observed in the present study, there was also an increased incidence of apoptosis and no net increase in the incidence of cancer. If MRL mice and p21 knockout mice are a good model for tissue regeneration in humans, temporary inactivation of the p21 gene could eventually be used to speed up wound healing in people.

The study is published in PNAS.

Controlling Fat Distribution

Extreme accumulation of fat in muscle tissue is associated with cardiovascular disease and is a contributory factor in insulin resistance and type II diabetes. It is therefore important to understand the mechanisms by which fat is taken up from the bloodstream and metabolised by tissues. Surprisingly, the role of blood vessels themselves in the transport of lipids has not been clearly established. Researchers at the Karolinska Institutet have now identified a role for vascular endothelial growth factor-B (VEGF-B) in endothelial targeting of lipids to peripheral tissues.

The VEGFs and their receptors are major regulators of angiogenesis and pharmacological intervention, for example with bevacizumab (a monoclonal antibody specific for VEGF-A), has been successfully exploited in oncology. This latest study has shown that VEGF-B, in mice, controls endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. Mice that were deficient in VEGF-B (Vegfb^-/-) showed reduced uptake and accumulation of lipids in muscle, heart and brown adipose tissue. Instead, the Vegfb^-/- mice preferentially transported lipids to white adipose tissue, resulting in a small weight increase. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium.

The authors of the study, published in Nature, propose that this new role for VEGF-B could potentially lead to novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Don’t Ignore the Neighbours

Out of every hundred investigational cancer drugs that enter clinical trials, only eight prove to be sufficiently safe and effective for FDA approval. Scientists at the Dana-Faber Cancer Institute believe that those odds can be shortened by taking into account neighbouring cells in the tumour microenvironment in preclinical testing.

Although both non-malignant cells and secreted proteins from tumour and stromal cells are recognised to be active participants in cancer progression, early laboratory testing is typically carried out using homogeneous populations of tumour cells. The Dana-Faber group have now developed a tumour cell-specific in vitro bioluminescence imaging (CS-BLI) assay that is suitable for high throughput screening. Cell viability can be measured in tumour cells stably expressing luciferase in the presence or absence of non-malignant accessory cells (for example, stromal cells) or drug treatment.

Unlike conventional screening methods, CS-BLI was able to identify compounds with increased activity against tumour cells interacting with stroma. For example, reversine showed more potent activity in an orthotopic model of diffuse myeloma bone lesions than in conventional subcutaneous xenografts. CS-BLI was also able to identify stroma-induced chemo-resistance such as imatinib resistance in leukaemia cells. The use of CS-BLI should help to identify preclinical candidates that overcome stroma-mediated drug resistance as well as those that can act in a synthetic lethal manner in the context of tumour-stroma interactions, thereby increasing the chances of clinical success.

The study is published in Nature Methods.

Role for Tau in ‘Microtentacles’ and Metastasis

Tau proteins interact with tubulin to stabilise microtubules and are abundant in neurons but less common in non-neuronal cells. Recently, tau proteins have received a bad press because of the association between tangles of hyperphosphorylated tau and Alzheimer’s disease and now researchers at the University of Maryland have reported that tau proteins may also play a role in tumour metastasis. Tau was found to promote formation of extensions of the plasma membrane or ‘microtentacles’ on breast cancer cells which break away from the primary tumour and circulate in the bloodstream. The microtentacles then increase the ability of the cells to attach to the walls of capillaries in the lung and seed new tumours. Tau protein has previously been associated with chemotherapy-resistant breast cancers and a poor prognosis but this is the first time that it has been implicated in metastasis. In the present study involving 102 breast cancer patients, 52% showed tau expression in metastases and 26% showed significantly increased tau expression as the disease progressed.

The team hope that drugs might be identified which will prevent the growth of microtentacles and inhibit tumour metastasis. Although current breast cancer treatments are often effective in treating the primary tumour, they are less effective in treating metastatic cancer which can develop years after the primary tumour is discovered and is the leading cause of death in cancer patients.

The study is published in the journal Oncogene.