The main symptoms of Parkinson’s disease are tremor, rigidity and involuntary movement, caused by loss of dopaminergic neurons in the brain. Leucine-rich repeat protein kinase-2 (LRRK2) is mutated in a significant number of Parkinson’s disease cases, both familial and sporadic late-onset. A common mutation in which a glycine residue in the active site is altered to serine enhances catalytic activity of the kinase, suggesting that LRRK2 inhibitors might be useful for the treatment of Parkinson’s disease, although it is not entirely clear why enhanced LRRK2 activity causes loss of dopamine-producing neurons. Scientists led by a team at the Johns Hopkins University School of Medicine have now shown that inhibitors of the G2019S variant of LRRK2 can protect the nerve cells of mice genetically modified to produce the mutated kinase. Three weeks twice daily injections of GW5074 provided almost complete protection against loss of dopaminergic neurons compared with placebo treatment.

Although GW5074 is not an especially potent inhibitor of wild type or G2019S LRRK2 (IC₅₀ 0.2µM – 1.0µM depending on substrate) and is not selective (IC₅₀ vs cRaf ca 10nM), the study, which is published in Nature Medicine, provides encouragement that a more potent and selective inhibitor could lead to a new disease-modifying treatment for Parkinson’s disease. The John Hopkins team are collaborating with researchers at Southern Methodist University to design more selective inhibitors and many other groups in both industry and academia are engaged in the search for potent and selective LRRK2 inhibitors.

The cytoskeleton plays a key role in regulating many cellular functions; it maintains cell shape, protects the cell, enables cellular motion, and has important roles in proliferation and differentiation. Metastasising cancer cells exploit the cytoskeleton to produce protrusions that allow them to invade surrounding tissue and enter the blood system from where they can spread to distant tissues and seed new tumours.

The protrusions, known as pseudopodia, are highly specialised ‘feet’ that the cell uses to pull itself forward across the underlying surface. A team led by researchers at the University of California, San Diego has now identified a previously unknown kinase – termed pseudopodium-enriched atypical kinase one or PEAK1 – that regulates the cytoskeleton and plays a central role in the formation of pseudopodia. Preliminary studies in mice suggest that PEAK1 is important during tumour growth and the team also showed that PEAK1 levels are increased in primary and metastatic samples from human colon cancer patients. Whether PEAK1 is capable of transforming non-tumour cells into cancer cells has not yet been determined but the fact that PEAK1 has kinase activity suggests that it may be possible to design specific inhibitors which could help to elucidate its role in both normal and cancer cells. PEAK1, which is a 190-kDa non-receptor tyrosine kinase, could serve as a clinical biomarker that predicts whether a cancer is likely to metastasise and could also be a target for future cancer treatments.

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

Apicomplexan parasites such as Toxoplasma gondii and Plasmodium species can cause serious diseases in humans and domestic animals. Because the parasites are eukaryotes and share many metabolic pathways with their hosts, it has proved difficult to develop safe and effective treatments but researchers at Washington University School of Medicine in St. Louis have now identified an essential kinase in T. gondii which is unlike human kinases and more closely resembles those found in plants. In a study published in Nature, the team used conditional suppression to show that T. gondii calcium-dependent protein kinase 1 (TgCDPK1) is essential for survival of the parasite. The enzyme controls the ability of T. gondii parasites to secrete microneme proteins which allow the parasites to control their movement and move in and out of host cells.

It should be possible to exploit the differences between the parasite kinase and human kinases to develop potent and selective inhibitors of the parasite enzyme and the team have already identified compounds that block CDPK1 signalling without affecting human cells. Pyrazolopyrimidine-derived compounds such as 3-MB-PPI were found to specifically inhibit TgCDPK1 and disrupt the parasite’s life cycle at stages dependent on microneme secretion. The disruption was dependent on TgCDPK1 inhibition since parasites expressing a mutant kinase not sensitive to the inhibitors.

Calcium-dependent protein kinases have a kinase domain similar to that of calmodulin-dependent kinase, regulated by a calcium-binding domain in the C terminus. X-ray structures of TgCDPK1, published in Nature Structural and Molecular Biology, showed that, in the auto-inhibited (apo) form, the C-terminal activation domain resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as calmodulin-dependent kinase II. Calcium binding triggers reorganization of the C-terminal activation domain into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

CDPK1 may play a similar role in Plasmodium species which cause malaria, but the researchers predict that it may be harder to selectively inhibit the Plasmodium enzymes.

Plasmodium parasites, responsible for malaria in humans, have a complex lifecycle that is dependent on mosquito and human hosts. In human blood, the merozoite stage of the parasite invades red blood cells (erythrocytes), growing and multiplying before rupturing the cell and escaping to infect other erythrocytes. It is this profound effect on erythrocytes that is responsible for the symptoms of malaria – fevers, chills and anaemia. Untreated, the disease can be fatal and drug resistance is an increasing problem. With up to half a billion people infected each year and nearly a million deaths, mostly in sub-Saharan Africa, there is an urgent need for new treatments.

Researchers at Harvard School of Public Health (HSPH) were attempting to identify the mechanism by which Plasmodium falciparum merozoites enter erthyrocytes, but instead found a parasite protein that is essential for escape from the cells. When the protein, P. falciparum calcium-dependent protein kinase (PfCDPK5), was suppressed the parasites were trapped in the host cell and unable to infect new cells. In further experiments the team showed that these merozoites were still able to invade erythrocytes if released from their host cell by other means, indicating separate mechanisms for invasion and egress from erythrocytes.

The findings reveal an essential step in the biology of P. falciparum and suggest a new, parasite-specific, drug target for fighting one of the world’s most common and dangerous infections. Whilst many scientists are looking for inhibitors of parasite egress and invasion of red blood cells, no anti-malarial drugs yet target these stages of the parasite lifecycle.

The study is published in Science.

The relatively rapid development of drug resistance is a major obstacle to successful chemotherapy. Resistance is frequently attributed to the outgrowth of cells within the tumour which have a genetic survival advantage in the presence of drug treatment such as enhanced drug efflux, impaired drug binding or the ability to use alternative survival pathways. More recently, it has been found that acquired drug resistance does not necessarily need a stable, heritable genetic alteration and, moreover, that response to treatment can be restored following a ‘drug holiday’. Whilst modelling the acute response to a variety of anti-cancer drugs in treatment-sensitive human tumour cell lines, researchers at Massachusetts General Hospital Cancer Center and the Dana-Farber Cancer Institute consistently found a small subpopulation of reversibly ‘drug-tolerant’ cells. They found that whereas the vast majority of EGFR mutant non-small cell lung cancer-derived cells (PC9 cells) were killed by exposure to a high concentration (100 x IC₅₀) of EGFR tyrosine kinase inhibitors (TKIs), a small fraction of cells survived. Similar populations of ‘drug-tolerant persisters’ (DTPs) were found when PC9 cells were treated with cisplatin and also in several other cancer cell lines with established drug sensitivity, suggesting that a drug-tolerant cell subpopulation is broadly present in tumour-derived cell lines.

Although DTPs are largely quiescent, about 20% eventually resume proliferation in the presence of drug to give colonies of cells referred to as ‘drug-tolerant expanded persisters’ (DTEPs) which can propagate indefinitely in the presence of drug. DTPs rapidly regain sensitivity when grown in drug-free media whereas restoration of sensitivity in DTEPs occurs at higher passage number. The reduced drug-sensitivity of both DTPs and DTEPs was linked to increased expression of a gene that encodes a chromatin-modifying enzyme, KDM5A. Although there are, as yet, no inhibitors of KDM5A, its known association with histone deacetylases (HDACs) led the team to test the effect of HDAC inhibitors on DTPs and DTEPs. Trichostatin A, an inhibitor of class I/II HDACs was found to rapidly kill PC9-derived DTPs and DTEPs but to have no effect on parental PC9 cells or TKI-resensitised DTEPs. The team went on to show that continous treatment with HDAC inhibitors, whilst having no effect on growth and survival of parental P9 cells, can prevent the emergence of EGFR TKI resistance. As well as HDAC inhibitors, a selective inhibitor of the insulin-like growth factor 1 receptor (IGF-1R) kinase also virtually eliminated the emergence of EGFR TKI-tolerant DTEPs. IGF-1R signalling was found to be necessary for drug-tolerant phenotypes in other cancer cell lines and to be mediated by the histone-demethylating activity of KDM5A.

The team hope that the results seen in cell culture experiments will extend to cancer patients and have already begun a clinical trial to see whether a combination of a chromatin-modifying agent with the EGFR TKI, erlotinib, may prevent or delay the development of resistance. Although the trial is not yet completed, early data indicate that the inclusion of a chromatin-modifying agent can dramatically improve clinical benefit in a subset of patients demonstrating acquired TKI resistance.

The study is published in the journal Cell.

Conventional wisdom, supported by in vitro experiments, has previously suggested that phosphoinositide 3-kinase (PI3K) plays a protective role in Alzheimer’s disease. However, a team led by researchers at Cold Spring Harbor has now implicated PI3K in the pathogenesis of the disorder.

The team used fruit-flies (Drosophila) that were engineered to produce human β-amyloid in their brains – a model that mimics many of the features of Alzheimer’s, including age-dependent memory loss, neurodegeneration, β-amyloid deposits and plaque formation. In the model, the presence of β-amyloid enhances long term depression (LTD), a process in which nerve signal transmission at particular synapses is depressed for an extended period. The research demonstrated that the enhanced LTD was a consequence of increased PI3K activity and could be abrogated by genetic silencing or pharmacological inhibition of PI3K.

PI3K inhibition restored LTD to a normal level, rescued β-amyloid peptide (Aβ)-induced memory loss and reduced β-amyloid deposits in the Drosophila brain. The data suggest that Aβ42 stimulates PI3K, which in turn causes memory loss in association with increased accumulation of Aβ42 aggregates.

The researchers note that the up-regulation of PI3K may also explain the insulin-resistance observed in the brains of Alzheimer’s victims. Insulin is one of the molecules that normally induce PI3K activity, which in turn mediates the cell’s response to insulin. Since PI3K is already hyperactivated in response to β-amyloid, it may no longer be able to respond to insulin.

The study is published in PNAS.

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.

Protein kinases play important roles in regulating most cellular processes and are commonly activated in cancer cells. A number of kinase inhibitors – including antibodies and small molecules –have already been approved for the treatment of cancer and many others are currently being tested. The majority of kinase inhibitors developed so far are ATP mimetics identified by high-throughput screening of catalytic kinase domains at low ATP concentration. Such compounds – so-called type I inhibitors – may lack specificity for individual kinases and/or be less effective when ATP concentrations are high. Crystal structures have revealed that some compounds – the type II inhibitors – occupy an allosteric site accessible only in the inactive conformation of the kinase and researchers at the Moores Cancer Center at the University of California have now designed selective type II inhibitors of PDGFRβ (important for pericyte recruitment) and B-RAF (important for endothelial cell survival).

Using the X-ray crystallographic structure of the type II inhibitor, sorafenib, bound to B-RAF, the team designed a small library of compounds based on a constrained amino-triazole scaffold predicted to stabilise kinases in the inactive state. The compounds were then tested for antivascular activity in both cell-based models and a zebrafish embryogenesis model. Compound 6 was found to inhibit both PDGFRβ and B-RAF cellular signalling – which produces a synergistic effect on tumour growth – but to have no effect on a variety of other cellular targets. The compound showed antiangiogenic activity in both zebrafish and murine models of angiogenesis and was also shown to suppress murine orthotopic tumors in both the kidney and pancreas.

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

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.