Drug Discovery Opinion

Fatty acids can be stored as triacylglycerol in lipid droplets, typically within adipose tissue, and then later released by the action of triacylglycerol hydrolase (TGH, also known as carboxylesterase-3, Ces3). Under normal circumstances, the released fatty acids provide an energy source, but excessive accumulation of triacylglycerol in peripheral tissues is associated with obesity and is a risk factor for type II diabetes and cardiovascular disease.

Researchers at the University of Alberta, Canada, reasoned that blocking the action of TGH would lead to better blood lipid profiles, but might also result in accumulation of triacylglycerol in the liver. However, they have found that mice lacking TGH (tgh^-/-) display global metabolic benefits with no obvious down-side. In both fasted- and fed-states, the animals had reduced plasma triacylglycerol, apolipoprotein B, and fatty acid levels. Despite the attenuation of very low-density lipoprotein (VLDL) secretion, TGH deficiency did not increase hepatic triacylglycerol levels. The tgh^-/- mice exhibited increased food intake and energy expenditure without change in body weight, and these metabolic changes are accompanied by improved insulin sensitivity and glucose tolerance.

The authors of the study, published in Cell Metabolism, suggest that pharmacological inhibition of TGH could be a useful therapeutic target, although cautioning that further work is required. It may be desirable to target TGH in specific tissues (e.g. hepatic versus adipose) but those subtleties have yet to be established.

Gene expression profiling is used to guide treatment options for women with breast cancer. Endocrine therapies – tamoxifen or aromatase inhibitors – are offered to women whose cancer is oestrogen receptor (ER) positive whilst the monoclonal antibody, trastuzumab (Herceptin®) and the small molecule, lapatinib (Tykerb®) are used to treat women whose cancer overexpresses the HER2 receptor. About 15% of breast cancers – the so-called triple negative breast cancers that don’t have receptors for oestrogen, progesterone or HER2 – don’t respond to hormone therapy or to HER2 blockers and the prognosis for women with these cancers is relatively poor.

Researchers at Washington University University School of Medicine in St. Louis have now identified a gene that is overexpressed mainly in ER-negative, HER2-negative and triple negative breast cancers, leading to the possibility of a new clinical biomarker and potential treatments. Upregulation of Wnt signalling coreceptor, LRP6 (low-density lipoprotein receptor-related protein 6), was found in about a quarter of the breast cancer samples that the researchers examined. Previous studies had shown that the protein Mesd (mesoderm development) blocks LRP6 and was able to slow the growth of cultured breast cancer cells. Mesd also inhibits the development of mammary tumours in mice, without producing known pathway-dependent side-effects such as bone lesions, skin disorders or intestinal malfunctions. A smaller fragment of Mesd was found to be as effective as full length Mesd and to have improved stability.

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

Although the study offers the prospect of targeted therapy for women with breast cancer that is currently difficult to treat, both screening and prescribing practices need to improve for such discoveries to realise their full potential. A recent news feature in Nature Biotechnology highlights differing views on testing as well as the problems associated with diagnostic tests for HER2 – both of which may be compromising women’s access to appropriate and effective treatment.

Cerebral cavernous malformations (CCM) are irregular clusters of dilated, leaky capillaries found in the central nervous system in around 0.5% of the general population. Although many of those with the condition will never be aware of the fact, for others the symptoms can be severe. Depending on the specific location of the CCM in the brain or spinal cord, patients may experience seizures, headaches, paralysis, hearing or vision changes, and cerebral haemorrhage. Current treatment options rely on management of the symptoms (e.g. control of seizures with anti-epileptic drugs) or surgical resection.

Researchers at University of North Carolina School of Medicine, Chapel Hill have now identified a potential target for therapeutic intervention in CCM. The disease is associated with mutations in any of three genes, ccm1, ccm2 or ccm3, which encode the corresponding CCM-1, -2 and -3 proteins. These proteins form a common complex and act co-ordinately in regulation of the cytoskeleton. It had previously been shown that loss of CCM-2 resulted in overexpression of the GTPase, RhoA, but this latest study demonstrates that CCM-1 and CCM-3 are also required for regulation of RhoA.

The team were able to restore normal function to endothelial cells lacking CCM-1, -2 or -3 by inhibition of the RhoA-activated Rho Kinase (ROCK), either using an inhibitor, Y-27632, or shRNA knockdown of ROCK2. The results suggest that inhibition of ROCK may represent a target for pharmacological intervention in this disease.

The study is published in the Journal of Biological Chemistry.

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.

The lifecycle of all Plasmodium species is complex and involves a round of replication in host erythrocytes. The clinical manifestations of malaria are linked to this stage in the lifecycle and are associated with rupture of the infected erythrocytes. During this growth phase, the parasite enters the erythrocyte and then releases several hundred effector proteins into the cytoplasm. These key virulence proteins provide a suitable environment for multiplication and allow the parasite to evade the host immune system. Proteins destined for export contain a conserved pentameric motif known as PEXEL and, when this is cleaved in the endoplasmic reticulum, the protein can be transported into the host cell. Two independent studies by scientists in the US and Australia have now shown that the protease responsible for cleaving the PEXEL motif is the aspartyl protease, plasmepsin V. Cleavage reveals an export signal at the amino terminus of the cargo protein which is then transported into the host cell cytoplasm, likely through a channel in the parasite’s outer membrane. Since export of the effector proteins is essential for the erythrocytic stage of the plasmodium life-cycle, drugs that block plasmepsin V should provide an effective treatment for malaria.

Both studies are published in the journal Nature (Australian study; US study).

The presence of multiple redundant and compensatory pathways controlling energy homeostasis has, so far, limited the effectiveness of anti-obesity treatments and suggests that combination therapy may be the best approach for treating the worldwide obesity epidemic. Writing in the journal Cell Metabolism, researchers at Merck have now demonstrated a role for the orphan bombesin receptor subtype 3 (BRS-3) in controlling energy balance.

Using a selective BRS-3 agonist (Bag-1) and antagonist (Bantag-1), the team have established a role for BRS-3 in the regulation of food intake, metabolic rate, and body weight. Intracerebroventricular infusion of the peptide Bantag-1 led to higher food intake and a progressive increase in adipose mass and body weight whereas oral administration of Bag-1 increased metabolic rate and reduced food intake, adipose weight, and body weight. Prolonged high levels of brain receptor occupancy by agonist increased weight loss, suggesting a lack of tachyphylaxis.

As well as suggesting a potential new target for the treatment of obesity, the discovery of selective BRS-3 agonists and antagonists will allow investigation of the mechanisms by which BRS-3 regulates energy metabolism as well as exploration of other aspects of BRS-3 biology.

An aortic aneurysm is a bulge in the aorta, the largest blood vessel in the body, which results from weakening of the artery wall. The majority of these occur in the portion of the aorta that passes through the abdomen and are referred to as abdominal aortic aneurysms (AAA). AAA is something of a stealth disease, since it is generally asymptomatic and may only be diagnosed at a routine physical examination or following X-ray. Over time the aneurysm may expand, with an increased risk of rupturing. Unfortunately, the rapid blood loss following aneurysm rupture is frequently fatal and accounts for at least 15,000 deaths in the US annually.

The only treatment for AAA currently available is surgical intervention. Early diagnosis is followed by monitoring the size of the aneurysm until the risk of rupture exceeds the risk of surgery. However, scientists at Providence Heart + Lung Institute at St. Paul’s Hospital and the University of British Columbia (UBC) have now raised the possibility of pharmacological intervention. Using experimental models of AAA, the team have found a role for the protein-degrading enzyme Granzyme B (GMZB).

GMZB is a serine protease expressed by a variety of immune cells and is responsible for destroying unwanted tissue, such as virally-infected cells. This role is supported by the pore-forming protein, perforin, which delivers GMZB to the intracellular compartment. The UBC research has shown that GMZB, which is abundantly expressed in aneurysms from human and animal model AAA, also plays a role in the pathogenesis of AAA. Further, the experimental data suggest that this is a perforin-independent mechanism involving extracellular matrix degradation and subsequent loss of vessel wall integrity. The results suggest that an inhibitor of GMZB may provide a therapeutic option in the treatment of AAA.

The study is published in the American Journal of Pathology.

Ischaemic stroke is a leading cause of adult disability and death. Glutamate plays an essential role in neural development, excitatory synaptic transmission, and plasticity but, during a stroke, glutamate accumulates at synapses, resulting in neuronal death. Excessive influx of Ca²⁺ ions through N-methyl-D-aspartate (NMDA) glutamate receptors is a major contributor to cell death and brain damage following ischaemic stroke.

So far, directly targeting glutamate receptors has not proved to be an effective way of treating stroke but scientists at the University of Central Florida and Louisiana State University have discovered that uncoupling a kinase from the NR2B subunit of the NMDA receptor blocks damaging Ca²⁺ influx through the receptor channels and protects neurons against the harmful effects of ischemia. Death-associated protein kinase 1 (DAPK1) is recruited into the NMDA receptor NR2B protein complex during ischaemia and phosphorylates NR2B at Ser-1303, enhancing the NR1/NR2B channel conductance. Genetic deletion of DAPK1 or administration of the peptide, NR2BCT – which blocks the interaction of DAPK1 with the NR2B subunit – protected mice from the damaging effects of cerebral ischaemia. NR2BCT did not affect the catalytic activity of DAPK-1 or the normal physiological functioning of NMDA receptors and the authors hope that, as well as providing new insights into the mechanisms of stroke damage, their discovery will provide a new target for the treatment of stroke which could show advantage over NMDA antagonists.

The study is published in the January 22^nd issue of Cell.

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer’s disease (AD) and correlate strongly with synaptic loss and severity of dementia. NFT appear to be attributable, at least in part, to hyperphosphorylation of the microtubule-stabilising protein, tau. Numerous phosphorylation sites have been associated with tau dysfunction and neurodegeneration: phosphorylation on Ser²⁶² has been shown to occur early in disease progression and to significantly reduce the affinity of tau for microtubules. Although hyperphosporylation at other sites is likely necessary for neurodegeneration, increased phosphorylation at Ser²⁶² is an important early step and firm identification of the kinase(s) responsible for phosphorylation at this position could provide new targets for disease-modifying treatments. Numerous kinases have been reported to phosphorylate tau at Ser²⁶² in vitro, but the role of these kinases on neurofibrillary tangle formation in vivo remains unclear.

Using a loss of function high throughput RNAi approach to screen the entire human kinome, a team led by scientists at the Translational Genomics Research Institute (TGen) have now identified three new kinases, dual specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), A-kinase anchor protein 13 (AKAP13), and eukaryotic translation initiation factor 2-alpha kinase 2 (EIF2AK2) that contribute to hyperphosphorylation of tau. Whereas DYRK1A and AKAP13 appear to be specifically involved in tau phosphorylation pathways, the effects of EIF2AK2 may result from alterations in tau protein expression.

If further studies in neuronal cell lines and in vivo models of AD and tauopathies confirm the importance of these kinases in disease pathology, they would represent novel targets for disease-modifying treatments for AD.

The study is published in BMC Genomics.

Cancer stem cells (CSCs) have been identified in a variety of tumour types, including breast tumours, and have been proposed to be responsible for recurrence, resistance to chemotherapy and, perhaps, metastasis of cancers. Targeting of these CSCs in the treatment of cancer is therefore of great interest. The ability of the ionophore antibiotic, salinomycin, to kill breast tumour CSCs was recently reported and now collaborators from the University of Michigan Comprehensive Cancer Center and the Centre de Recherche en Cancérologie de Marseille have identified a new potential drug target.

In this latest study the team identified overexpression of CXCR1, the receptor for interleukin-8 (IL-8), by the CSC subpopulation in a breast cancer cell line. Furthermore, addition of recombinant IL-8 increased the CSC population and enhanced the propensity for invasion. Conversely, use of CXCR1-blocking antibodies or repertaxin, a small-molecule CXCR1 antagonist, selectively depleted the breast CSCs both in vitro and in murine xenograft models.

CXCR1 blockade also induced massive apoptosis in bulk tumour cells, mediated by FASL/FAS signalling. The effects on CSC viability as well as FASL production were mediated by the focal adhesion kinase/AKT/forkhead transcription factor FKHRL1 (FAK/AKT/FOXO3A) pathway. Importantly, administration of repertaxin reduced tumour growth and the development of systemic breast cancer metastasis in NOD/SCID mice.

The authors of the study, published in the Journal of Clinical Investigation, suggest blockade of CXCR1 as a novel target for depletion of CSCs, potentially enhancing the efficacy of chemotherapeutic regimes.