Rational Design of New Type II Kinase Inhibitor

Crystal structure of sorafenib complexed with B-RAF, PDB ID=1UWH
Crystal structure of sorafenib complexed with B-RAF, PDB ID=1UWH
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).
compound 6
Compound 6

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.

Role for Death Protein in Stroke

Image: Flickr - Bixentro
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 22nd issue of Cell.

Three New Kinase Targets for Alzheimer’s Disease

Image: Wikimedia Commons - Childzy
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.

Mapping Phosphorylation in Cancer

Phosphorylation is a key mechanism for regulation of protein activity and the phosphorylation of tyrosine residues, in particular, is important in signalling pathways. Aberrant phosphorylation has been observed in many cancers and has driven the development of kinase inhibitors that have utility in a number of cancer subtypes. Since abnormal activation of signalling pathways is a common feature that accompanies tumour initiation and progression, methods to assess signalling pathway status of cancer tissue relative to normal could provide important insights.

A group of US researchers has now conducted a detailed analysis of tyrosine phosphorylation states in lung cancer compared to normal tissue. The work documents a large set of sites that are differentially phosphorylated in the cancer tissue, immediately providing a number of drug target candidates. Taking the study a step further, the group has developed computational methodology to identify signalling pathways where phosphorylation activity is strongly correlated with the lung cancer phenotype.

The EGFR signalling pathway. Pink indicates higher phosphorylation in tumour samples, while green indicates higher phosphorylation in normal tissue samples. Full details can be found in PLoSone.
The EGFR signalling pathway. Pink indicates higher phosphorylation in tumour samples, while green indicates higher phosphorylation in normal tissue samples. Full details can be found in PLoSone.

The results provide a highly predictive set of signatures that reliably distinguish each lung cancer from normal. Since the signatures identify proteins and pathways where phosphorylation should be inhibited, the methodology highlights potential new targets for drug discovery.

The study is published in the journal PLoSone.

Suppressing ROCK May Prevent Breast Cancer Metastases

Image: Flickr - Austin UK
Image: Flickr - Austin UK
Nearly half of all patients treated for apparently localised breast cancer develop metastatic disease. Although there are treatment options that prolong survival and improve quality of life for patients with metastatic breast cancer, these treatments rarely lead to long-term survival without disease recurrence. The most common sites of metastases are the bones, the liver and the lungs: breast cancer is the most common origin of metastatic deposits in the skeleton.

Researchers from Tufts University have now identified Rho-associated kinase (ROCK) as a potential target to reduce breast cancer metastasis. Signalling through ROCK plays a key role in regulating cell adhesion and motility and aberrant expression of ROCK has been linked to metastasis. In the present study, the team showed that ROCK expression is increased in metastatic human mammary tumours and breast cancer cell lines compared with non-metastatic tumours and cell lines. A metastatic phenotype could also be induced in a cell line that is not normally metastatic by over-expression of ROCK.

In a novel mouse model of “human breast cancer metastasis to human bone”, inhibiting ROCK in the earliest stages of breast cancer decreased metastatic tumour mass in bone by 77% and overall frequency of metastasis by 36%. ROCK function could be effectively blocked by either ROCK-targeting short hairpin RNA (shRNA) or by the specific ROCK inhibitor, Y27632. Expression of the microRNA cluster, c-Myc-regulated miR-17-92 was found to be elevated in metastatic breast cancer cells compared with non-metastatic cells and reduced by treatment with Y27632. Blockade of miR-17 was further shown to decrease breast cancer cell invasion/migration in vitro and metastasis in vivo. The authors suggest that the effects of ROCK may be mediated by modulating the c-Myc pathway, including c-Myc-dependent microRNAs. They propose that inhibition of ROCK, or the pathway it stimulates, may represent a novel approach for treatment of breast cancer metastases.

The study is published in the journal Cancer Research.

Extracurricular Role for ERK2 as a Transcription Factor

spiral structure
Image: Flickr - Andrew Stawarz
Scientists at Johns Hopkins School of Medicine set out to systematically map protein-DNA interactions across the human genome using a combination of bioinformatics and a protein-microarray strategy. Their exploration of the protein-DNA interactome identified over 17,000 interactions between 460 DNA sequences predicted to regulate transcription and 4191 proteins of varied functional classes. As well as finding known transcription factors (TFs), the work uncovered a large number of previously uncharacterised TFs. Of the unconventional DNA-binding proteins, over 300 exhibited sequence-specific DNA-binding.

One example of the sequence-specific DNA binders identified was ERK2 (MAP kinase-1), a serine/threonine kinase involved in cellular signalling. The Johns Hopkins team found that ERK2 is a transcriptional repressor of interferon-γ inducible genes and that this function is independent of its catalytic kinase activity. Whilst ERK2 has been extensively studied because of its importance in regulation of cellular proliferation, this unexpected additional role of ERK2 adds another level of complexity. Knowledge of the transcriptional repressor function may shed new light on knock-out experiments with ERK2.

The study, published in the journal Cell, examined only a fraction of the human proteome, raising the possibility that there may be thousands of proteins that also function as transcription factors. Certainly something else to consider in drug discovery when your enzyme inhibitor doesn’t do the same as the knockout!

Syk Protective Against Breast Cancer

Inhibitors of the non-receptor spleen tyrosine kinase (Syk) are being developed to treat a variety of allergic and autoimmune disorders, as well as some types of cancer, but researchers at Georgetown University Medical Center have cautioned that Syk controls the growth of normal breast cells and prevents the development of breast cancer.

Although it is not known what causes loss of Syk function, Syk is negatively correlated with invasion and metastasis of tumour cells and, as breast tumours progress, more and more Syk protein is lost. Since total knock-out of Syk is perinatally lethal, the researchers created mice with only one copy of the gene and found that loss of the single allele led to increased proliferation and invasion of normal breast cells in the mouse mammary gland during puberty, and resulted in development of breast cancer in adulthood.

Three colour confocal images of cells cultured on crosslinked gelatin showing the distribution of DAPI (nuclei, blue), phalloidin (F-act, green), and vimentin (magenta) in control (left) versus Syk siRNA transfected MCF10A cells (right). Scale bars = 20 µm. Image: PLoSone
Three colour confocal images of cells cultured on crosslinked gelatin showing the distribution of DAPI (nuclei, blue), phalloidin (F-act, green), and vimentin (magenta) in control (left) versus Syk siRNA transfected MCF10A cells (right). Scale bars = 20 µm.

Image: PLoSone

siRNA or shRNA knockdown of Syk protein in cultures of normal human breast epithelial cells also dramatically increased proliferation and invasion. Syk loss was shown to release inhibition of a number of signalling pathways that are normally repressed in epithelial cells and that promote increased proliferation, motility, and invasiveness. The findings, which were published on October 15th in the journal PLoS ONE, suggest that Syk plays an important role in controlling growth as breast tissue develops and acts as a tumour suppressor for breast cancer. The finding that only partial loss of Syk function in mice was sufficient to induce mammary carcinomas underscores the potential risk of Syk inhibition in promoting breast cancer. The team hope that identification of the pathways that are negatively regulated by Syk will ultimately provide new targets for the treatment of breast cancer.

GSK-3 – Master Controller of Neural Stem Cells

Image: Flickr - Khazaei
GSK-3 (glycogen synthase kinase-3) is a serine-threonine kinase that consists of two family members in mammals, α and β, which share 98% sequence identity in their kinase domains. The β-isoform has recently been identified as a mediator of neurogenesis under the control of DISC1, a protein encoded by a gene implicated in schizophrenia susceptibility. GSK-3 has also been shown to be involved in regulation of embryonic stem cell self-renewalGSK3 inhibitors help in maintenance of pluripotency.

A new study from researchers at the University of North Carolina at Chapel Hill School of Medicine has now shown that GSK-3 is a key regulator of neural stem cell proliferation and differentiation. Neural stem cells progress through different stages – neural epithelial cells, radial progenitor cells and intermediate neural precursors – and radial progenitor cells are particularly important because they are thought to provide the majority of the neurons of the developing brain and to give rise to all the cellular elements of the brain. The researchers used a conditional knockout in a mouse model, deleting both isoforms of GSK-3 during the radial progenitor phase of development. This resulted in locking the radial progenitor cells in a proliferative state, with no generation of mature neurons. The next step is to determine whether switching GSK-3 back on can stimulate differentiation, leading to an increased number of mature neurons. The researchers suggest that understanding the role of GSK-3 in neurogenesis could have implications for patients with neuropsychiatric conditions such as schizophrenia, depression and bipolar disorder.

The study is published in the journal Nature Neuroscience.

New Target for Malignant Melanoma

Sun photo by Radiant Guy
Photo: Radiant Guy
Malignant melanoma is the most aggressive form of skin cancer, involving malignant transformation of melanocytes. Although it is one of the less common types of skin cancer, it accounts for around 75% of skin cancer-related deaths. The greatest chance of survival currently depends on early diagnosis and surgical removal of the tumour.

Melanocyte differentiation is under the control of microphthalmia-associated transcription factor (MITF), a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. Paradoxically, however, high expression of MITF also has an anti-proliferative effect. Scientists at Keio University School of Medicine, Shinjuku-ku, Tokyo and collaborators have demonstrated that both depletion and forced expression of MITF in human melanoma cell lines significantly inhibited proliferation. Although approximately half of the cell lines were resistant to MITF depletion, simultaneous depletion of both MITF and BRAFV600E, an oncogenic kinase also associated with melanoma, significantly inhibited melanoma growth even in the resistant cell lines. The work, published in the October edition of Cancer Science, suggests that dual inhibition of MITF and BRAFV600E represents a useful strategy for treatment of melanoma.

A second study, from collaborators at The Scripps Research Institute and the Genomics Institute of the Novartis Research Foundation, has found that the receptor tyrosine kinase, TYRO3, is an upstream regulator of MITF expression. TYRO3 expression is significantly elevated in human primary melanoma tissue samples and melanoma cell lines and correlates with MITF mRNA levels. Their study showed that TYRO3 overexpression bypassed BRAFV600E-induced senescence in primary melanocytes, inducing transformation of non-tumourigenic cell lines. Knockdown of TYRO3 repressed cellular proliferation of melanoma cells and sensitised them to chemotherapy-induced apoptosis. In addition, TYRO3 knockdown in melanoma cells also inhibited tumourigenesis in vivo. The study is published in the 23rd September online edition of PNAS.

Taken together, the two studies suggest that a dual TYRO3/ BRAFV600E inhibitor would be interesting to evaluate in melanoma.

Not Channelling for Success

canal lockGlobally, hepatitis C virus (HCV) infects almost 200 million people and is a leading cause of cirrhosis and hepatocellular carcinoma – albeit several decades after initial infection. In a majority of cases, the virus is able to establish a persistent infection and, even with current gold standard treatments, sustained cure rates once the infection has become established are only around 50%. Researchers at the University of Leeds have now uncovered a previously unrecognized mechanism that allows the virus to evade the immune system and establish a chronic infection.

Establishment of persistent infection means that HCV-infected cells must be resistant to pro-apoptotic stimuli and the team found that one of the viral proteins, NS5A, is able to block apoptosis in human hepatoma cells either infected with HCV or harbouring an HCV subgenomic replicon. Amplification of an outward K+ current mediated by voltage-gated Kv2.1 channels normally precedes apoptosis triggered by oxidative stress and NS5A was found to block this process by inhibiting phosphorylation of Kv2.1 by p38 MAP kinase. Inhibition of a host cell ion channel by a viral protein as a means of preventing apoptosis has not previously been described, and the researchers hope that their findings could lead to new strategies for antiviral therapy. The study is published in the August 26th online edition of PNAS.