Role for c-Abl in Parkinson’s Disease

Shaking Man
Image: Flickr - bearroast
The majority of Parkinson’s disease (PD) cases have no known cause, but have been associated with increased oxidative stress and mitochondrial dysfunction. Of the small proportion of hereditary cases, a number of defective genes have been identified including LRRK2 (PARK8), DJ-1 (PARK7), α-synuclein (SNCA) and parkin (PARK2). Mutations in parkin, which encodes an E3 ubiquitin ligase, are believed to interfere with the ability of parkin to clear the cell of its normal substrate proteins. Several substrates for parkin have been identified and shown to accumulate in the brain tissue of patients with hereditary PD.

Researchers at The University of Texas Health Science Center have now identified a link between the tyrosine kinase, c-Abl, and impaired parkin function. The scientists found that c-Abl was activated in cultured neuronal cells and the striatum of adult mice when subjected to oxidative and neuronal stress. They also identified parkin as a specific substrate for c-Abl and that the tyrosine-phosphorylated parkin lost its ubiquitin ligase activity.

The c-Abl inhibitor, imatinib (STI-571) was able to block the phosphorylation of parkin in vitro and in vivo, restoring ligase activity. Since there are several c-Abl inhibitors approved for the treatment of chronic myelogenous leukemia, tools are available to further explore the neuroprotective potential of c-Abl inhibition in sporadic PD.

The study is published in the Journal of Neuroscience.

A Histidine Kinase as a Target for Autoimmune Diseases

NDPK-B crystal structure
Crystal structure of the hexameric NDPK-B protein - PDB ID=3BBB
The nucleoside diphosphate kinases (NDPK) comprise a family of 10 members encoded by the Nme (non-metatstatic cell) gene family. These kinases are capable of transferring the γ-phosphate of nucleoside triphosphates to nucleoside diphosphates, which is accomplished via a phospho-histidine intermediate. Since their discovery, the NDPKs have been shown to play a role in numerous cellular processes. Of the 10 members, NDPK-A and B (also known as NM23-H1 and NM23-H2 respectively) are ubiquitously expressed and account for >95% of NDPK activity in most cells.

NDPK-A and B regulate cellular processes through a variety of mechanisms including generation of nucleoside triphosphates, histidine phosphorylation, protein-protein interactions and regulation of downstream signalling pathways. Interestingly, the NDPKs are currently the only known histidine kinases found in mammals.

Despite sharing 88% sequence identity, NDPK-A and B have each been associated with specific functions. Nevertheless, there appears to be significant redundancy within the family. NDPK-A knockout mice have been reported to be phenotypically normal, with the exception of reduced birth weight and delayed mammary development. However, double knockout of NDPK-A and B results in stunted mice that die perinatally as a result of severe anaemia and abnormal erythroid development.

Now a team at New York University Medical Center have reported the mouse knockout of NDPK-B. Previously the team had shown that NDPK-B activates the K+ channel, KCa3.1, by phosphorylation of 358His in the KCa3.1 carboxy terminus. Since this activation is required for T-cell receptor stimulation of Ca2+ flux and proliferation of naïve human CD4+ T-cells, the team speculated that inhibition of NDPK-B could represent a target for therapy of autoimmune diseases.

The NDPK-B knockout mice were phenotypically normal at birth, with normal T and B cell development. KCa3.1 channel activity and cytokine production were defective in Th1 and Th2 cells (but normal in Th17 cells), however, confirming the importance of NDPK-B in T cell activation. The data support the concept of NDPK-B inhibition as a therapeutic strategy, although specificity for NDPK-B over the A isoform will be necessary. Given the degree of sequence conservation between the two isoforms, this could be a significant challenge.

The BMK1 Pathway in Oncology – a Road Less Travelled

Image: Flickr – Zach Chisholm
Of the four mammalian MAP kinase pathways (ERK1/2, JNK, p38 and BMK1), BMK1 is the least studied. BMK1 and ERK1/2 pathways are both activated by mitogens and oncogenic signals and are therefore implicated in tumorigenesis. Indeed, the ERK1/2 pathway has received significant attention for the development of chemotherapeutic drugs. Deregulated BMK1 activity has been associated with a variety of human malignancies including chemoresistance of breast tumours, metastasis of prostate tumour cells and tumour-associated angiogenesis. Conditional knockout of endothelial BMK1 in mice, however, led to lethal vascular instability, discouraging exploration of BMK1 as a therapeutic target.

XMD8-92 structure
A new study from scientists at the Scripps Research Institute has revealed more detail on the role of BMK1 in oncogenesis and suggests that BMK1 inhibition could be a viable therapeutic strategy. The study found that BMK1 is associated with the tumour suppressor, PML (promyelocytic leukemia protein), and suppresses its anti-cancer activity. In cellular studies, reduced expression of BMK1 resulted in induced expression of p21, a downstream effector of PML and modulator of cell proliferation.

The team’s serendipitous discovery of a selective inhibitor of BMK1, XMD8-92, permitted further studies in animal models. XMD8-92 significantly inhibited the growth of xenografted human tumours in mice, with no obvious adverse effects. More specifically, in contrast to the BMK1 conditional knockout studies, no vascular instability was observed in response to pharmacological inhibition of BMK1.

The study is published in Cancer Cell.

LRRK2 Inhibitor Protective in Parkinson’s Disease Model

"Moondance" photographed by Tena (Madmoiselle Lavender), a Parkinson's veteran
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.

GW5074 structure
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.

Role for PEAK1 Kinase in Tumour Metastasis

Image: Flickr - Alaskan Dude
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.

Kinase Target for Toxoplasmosis, maybe Malaria

Crystal structure of inactivated form of CDPK1 from toxoplasma gondii
Crystal structure of inactivated form of CDPK1 from toxoplasma gondii - PDB ID 3KU2
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.

3-MB-PPI structure
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.

Locking out or Locking in – It’s Not the Same Key for Malaria

Image: Flickr – Svadilfari
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.

Reversing the Tide of Resistance

Image: Flickr - Joanna Vaughan
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.

Challenging the Role of PI3 Kinase in Alzheimer’s

Image: Flickr – Richard0
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.

Role for ROCK in Cavernous Malformation

Image: Flickr – Eschipul
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.

Y-27632 structure
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.