Blog – Page 3 – Drug Discovery Opinion

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.

One Step Nearer to Taxol

taxadien-5-alpha-ol Although Taxol® (paclitaxel) offers significant benefits to cancer patients, its initial isolation from the bark of the Pacific yew tree (Taxus brevifolia) raised serious ecological concerns since the trees are killed in the harvesting process. These concerns led to the development of both synthetic and semisynthetic routes to paclitaxel, but the drug is now manufactured more efficiently using plant cell cultures.

Scientists at MIT and Tufts University have now engineered a new strain of E. coli bacteria that can produce taxadiene and taxadien-5-α-ol, key precursors to paclitaxel. Although E. coli does not naturally produce taxadiene, it does produce isopentenyl pyrophosphate (IPP), a compound that is two steps back in the plant biosynthetic pathway. The team identified four bottlenecks in the eight-step E. coli biosynthesis of IPP and engineered the bacteria to produce multiple copies of the genes encoding the enzymes responsible for carrying out these four steps. To enable the bacteria to carry out the two additional steps needed to covert IPP to taxadiene, the researchers added the plant genes for the appropriate enzymes. By altering the copy numbers of genes to find the most efficient combination, the team were able to produce a strain of E. coli that produces more than 1000 times more taxadiene (ca 1g/L) than any other engineered strain. They then added an extra step towards the synthesis of paclitaxel, achieving the first microbial conversion of taxadiene to taxadien-5-α-ol.

There are another fifteen to twenty steps to go to achieve a microbial synthesis of pactitaxel but, if these can be achieved, as well as producing pactitaxel, the engineered bacteria should allow access to a variety of terpenoid natural products.

The study is published in the journal Science.

Blocking Cancer Networks

spider web — Image: Flickr - Luc Viatour

The emergence of drug resistance is one of the main causes of failure in cancer treatment and is one reason that cancer drugs are often used in combination. Resistance can also arise during the use of combinations of cytotoxic agents developed by trial and error but researchers at Fox Chase Cancer Center and Georgetown University have now developed a better way of selecting drug combinations based on molecular targets.

The epidermal growth factor receptor (EGFR) is a well validated molecular target and inhibitors are already used clinically for certain types of cancer. The team used siRNA to silence 638 genes known to encode proteins involved in the EGFR signalling network and identified over 60 proteins that can rescue cells in the presence of an EGFR inhibitor. Amongst these were three proteins for which drugs are already being developed: Aurora kinase A, protein kinase C, and STAT3. Aurora kinase A inhibitors are already being evaluated clinically and a trial testing the EGFR inhibitor erlotinib with an Aurora kinase inhibitor in patients with non-small cell lung cancer is being launched. A similar network-centred approach could be used to design other combination therapies to overcome resistance mechanisms in cancer.

Interestingly, the screen did not pick out genes previously linked to resistance to EGFR inhibitors and most of the genes identified were not mutated: KRAS mutations did not appear to be needed for resistance to EGFR inhibitors although patients with KRAS mutations do not benefit from EGFR inhibitors.

The study is published in Science Signaling.

Study Casts New Light on Prozac Mechanism

Selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine (Prozac®) have been used to treat depression for more than three decades but researchers from INSERM and Hoffmann-La Roche have now shed new light on their mechanism of action. SSRIs are believed to act by inhibiting uptake of serotonin into presynaptic cells, thereby increasing the amount available in the synapse to bind to postsynaptic receptors. Typically, an ‘adaptation phase’ of several weeks is needed before the antidepressant effects are fully manifest and the new study helps to explain this latency. The study identified a key role for microRNA-16 (miR-16) in regulating expression of the serotonin transporter (SERT) which is responsible for the recapture of serotonin. Under normal conditions, SERT is present in serotonergic neurons where levels of miR-16 are low, but expression is silenced in noradrenergic cells by higher levels of miR-16; a reduction of miR-16 in noradrenergic cells causes de novo SERT synthesis.

In mice, chronic treatment with fluoxetine was shown to increase levels of miR-16 in serotonergic cells, leading to reduced SERT expression. The cells also released the neurotrophic factor S100β, which decreased miR-16 in noradrenergic cells, resulting in cells with a mixed phenotype that produced both noradrenaline and serotonin and which were sensitive to fluoxetine. Treatment with fluoxetine thus increases serotonin levels both by preventing reuptake by serotonergic neurons and by stimulating production by noradrenergic neurons through reduction of miR-16.

The study is published in the journal Science.

The BMK1 Pathway in Oncology – a Road Less Travelled

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.

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.

Possible Biomarker for COX-2 Cardiovascular Risk

Die on Checkerboard — Image: Flickr - Cristian N Mormoloc

Selective COX-2 inhibitors were developed to minimise the adverse gastrointestinal effects seen with conventional NSAIDs and have provided effective pain relief for millions of arthritis patients. Long-term, high dosage use of some COX-2 inhibitors, however, was found to be associated with an increased risk of heart attacks and strokes, resulting in drug withdrawals. A clearer understanding of the mechanisms underlying the cardiovascular effects associated with COX-2 inhibitors would allow better risk/benefit assessment and could possibly lead to the development of safer inhibitors.

Researchers from the University of California, Davis and Beijing University have now shown that, in mice, oral administration of rofecoxib for 3 months leads to a more than 120-fold increase in the regulatory lipid, 20-hydroxyeicosatetraenoic acid (20-HETE) which correlates with a significantly shorter tail bleeding time. Further studies suggested that inhibition of COX-2-mediated 20-HETE degradation by rofecoxib may, at least in part, explain the increase in blood levels and shortened bleeding time and may also contribute to the cardiovascular side effects seen with rofecoxib. Although the relative importance of COX-2 in the metabolism of 20-HETE in man has not yet been determined, if it proves to be as important as in mice, blood levels of 20-HETE may be a good predictor of which patients are at higher risk of heart attack or stroke.

The study is published in the journal PNAS.

Speeding the Cellular Waste Disposal System

Garbage Trucks — Image: Flickr - Michael Kohli

The ubiquitin-proteasome system (UPS) is a critical element of the cellular machinery, responsible for removing unwanted proteins. Target proteins, which may be misfolded, oxidised or simply no longer required, are marked for degradation by attachment of ubiquitin chains. The ubiquitinated proteins are then recognised by the proteasome and subjected to proteolytic cleavage. Failure of the UPS leads to a build up of damaged or misfolded proteins that may result in cellular toxicity.

Accumulation of misfolded proteins is a feature of a number of human disorders including Alzheimer’s, Parkinson’s and Creutzfeldt-Jakob diseases. Upregulation of the UPS is therefore of interest for potential therapy of these disorders.

Usp14 crystal structure — Complex of Usp14 (grey spheres) with ubiquitin-aldehydye (blue ribbon). PDB ID=2AYO

A team from Harvard Medical School has been investigating the role of Usp14, a de-ubiquitinating enzyme associated with the proteasome. They found that Usp14 is able to inhibit the proteasomal degradation of ubiquitin-protein conjugates both in vitro and in cells. A catalytically inactive variation of Usp14 had reduced inhibitory properties, suggesting that Usp14 mediates its effects by cleavage of ubiquitin from the substrate proteins.

The team identified a small molecule inhibitor of Usp14 using high-throughput screening and treatment of cultured cells with this compound enhanced the degradation of proteasome substrates associated with neurodegeneration. The compound also accelerated the degradation of oxidised proteins and improved cellular resistance to oxidative stress.

The study, published in Nature, sheds light on a poorly understood aspect of the UPS – the control of the speed of protein degradation. The authors suggest that inhibition of Usp14 may be a strategy to address a variety of human diseases where accumulation of aberrant proteins is a factor.

2-AG – Not Such Eternal Bliss?

Ananda at the first Buddhist council — Ananda at the First Buddhist Council - Image: Wikipedia - Sacca

The cannabinoid receptors, CB₁ and CB₂, were first identified as the receptors for the active components of Cannabis sativa. The endogenous ligands for the cannabinoid receptors, anandamide (from ananda, a Sanskrit word meaning ‘bliss’) and 2-arachidonoylglycerol (2-AG), exert most of their analgesic affects through binding to the main cannabinoid receptor in the brain, CB₁. Attempts to prepare CB₁ agonists that mimic the analgesic effects of the main component of cannabis, Δ9-tetrahydrocannabinol (THC) but have reduced potential for memory impairment, locomotor dysfunction, and possibly addiction have met with limited success and a recent alternative approach has been to devise ways of preventing the breakdown of anandamide and 2-AG. Signalling by the endocannabinoids is terminated by enzymatic hydrolysis which, for anandamide, is mediated by fatty acid amide hydrolase (FAAH) and, for 2-arachidonoylglycerol, by monoacylglycerol lipase (MAGL).

Inhibitors of both FAAH and MAGL produce analgesic effects but scientists at the Scripps Research Institute, Virginia Commonwealth University and the Medical College of Wisconsin have now shown that the effects of blocking MAGL are short-lived compared with the effects of blocking FAAH. Although a single injection of the MAGL inhibitor, JZL184, reduced pain in the mouse, after six consecutive daily injections, the effect disappeared. The chronically treated animals also became less sensitive to THC or the synthetic CB₁ agonist, WIN55,212-2, and showed characteristic drug withdrawal symptoms when treated with the CB₁ blocker, rimonabant.

Further tests showed that CB₁ receptors were down-regulated in some brain areas of mice treated for 6 days with JZL184. Genetic disruption of MAGL also resulted in elevated levels of 2-AG and desensitised the CB₁ signalling system, suggesting that chronic inhibition of MAGL may not provide effective analgesia. In contrast, chronic treatment with a FAAH inhibitor to boost anandamide levels did not lead to desensitisation of the CB₁ system.

Although the team do not yet understand why chronic administration of FAAH inhibitors and MAGL inhibitors should produce such strikingly different results, the study, which is published in Nature Neuroscience, suggests that complete blockade of MAGL may not provide effective analgesia over the longer term.

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.

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.

How Arthritis May Protect Against Alzheimer’s Disease

RA and Amyloid Epidemiological studies have suggested that either rheumatoid arthritis itself – or the anti-inflammatory drugs used to control it – are associated with a reduced risk of developing Alzheimer’s disease. Recent clinical trials with non-steroidal anti-inflammatory drugs (NSAIDs) have failed to show a benefit in patients with Alzheimer’s disease and researchers at the University of South Florida have now shown that it may be the disease itself that affords protection.

In a mouse model of Alzheimer’s disease, one of the cytokines that is elevated in patients with rheumatoid arthritis, granulocyte-macrophage colony-stimulating factor (GM-CSF), was shown to improve working memory and learning and to lead to an apparent increase in neural cell connections in the animals’ brains. GM-CSF also led to an accumulation of microglia in the brains of treated animals which was associated with a greater than 50% reduction in β-amyloid peptides. Recombinant human GM-CSF is currently approved to stimulate the production of white blood cells in some cancer patients and the new study, which is published in the Journal of Alzheimer’s Disease, suggests that GM-CSF could also be of benefit to Alzheimer’s patients.

The USF Health Byrd Alzheimer’s Institute plans to begin a pilot clinical trial later this year to investigate recombinant human GM-CSF in patients with mild or moderate Alzheimer’s disease.