target identification – Page 2 – 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.

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.

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.

Nickel Allergy Linked to Innate Immune Response

piercing — Image: Wikimedia Commons - Nicor

Nickel allergy is one of the most common causes of allergic contact dermatitis and a team led by researchers at the University of Giessen have now shown that the response is linked to activation of a single receptor, toll-like receptor 4 (TLR4).

The family of toll-like receptors normally recognizes structurally similar molecules derived from microbial pathogens and plays a key role in host defense. The team showed that nickel directly activated human, but not mouse, TLR4 and studies with mutant proteins showed that non-conserved histidine residues at positions 456 and 458 were necessary for activation of human TLR4. Wild type mice do not show an allergy to nickel but transgenic mice expressing human, rather than mouse, TLR4 could be efficiently sensitized to the metal. This is the first time that an inorganic substance has been shown to activate this innate immune pathway and, since histidines 456 and 458 are not essential for responses to microbial lipopolysaccharide, the authors suggest that site-specific inhibition of TLR4 could provide a potential strategy for treatment of nickel allergy, which would not compromise normal immune responses. Nickel allergy affects millions of people and is often associated with earrings and jewellery for other body piercings.

The study is published in the journal Nature Immunology.

Role for MMPs in Huntington’s Disease

Neuron — Microscope image of a neuron with inclusion (stained orange) caused by HD, image width 250µm. Source: Wikipedia

Huntington’s disease is an inherited neurodegenerative disorder associated with mutations in the huntingtin gene on human chromosome 4. Although the functions of normal huntingtin protein are not entirely clear, it is known that abnormal huntingtin (mutantHtt, or mHtt) – and especially small proteolytic fragments of the protein – are toxic to neurons, particularly those in the striatum and cortex. Previous studies into the cleavage of huntingtin have focussed on the role of the cysteine protease families of caspases and calpains, but scientists at the Buck Institute for Age Research have now discovered that a metalloprotease also cleaves huntingtin into highly toxic fragments.

The team used a set of small interfering RNA (siRNA) pools targeting the 514 known and predicted human protease genes to identify those proteases involved in the cleavage of huntingtin. Eleven proteases were found to alter huntingtin fragment accumulation, and knockdown of the nine which are expressed in striatal cells significantly reduced huntingtin-mediated striatal cell death in a cellular toxicity screen. Amongst the proteases associated with huntingtin cleavage were three metalloproteases, MMP-10, MMP-14 and MMP-23B. Subsequent experiments showed that only MMP-10 directly cleaves huntingtin, suggesting that knockdown of MMP-10 reduces toxicity by directly altering proteolysis of huntingtin whereas knockdown of MMP-14 or MMP-23B modulates toxicity indirectly through proteolysis of cytokines or components of the extracellular matrix. Whilst matrix metalloproteases are generally thought to be secreted as proenzymes which are processed to the active forms extracellularly, MMP-10 was found to be activated inside the cell and to co-localise with huntingtin, suggesting that cleavage may occur intracellularly.

The study, which is published in the journal Neuron, suggests a role for matrix metalloproteases in the progression of Huntington’s disease and that development of inhibitors of MMP-10 may be a useful therapeutic strategy.

Genetic Basis for Alopecia Areata

Alopecia areata is a type of hair loss that typically begins with one or more small bald patches on the scalp, beard area or elsewhere. The patches appear quite quickly and the hair may re-grow after a few months – or the condition may persist for several years with recurrences of patchy baldness and hair re-growth. The condition can also result in total loss of scalp hair (alopecia totalis) and, in a small number of cases, total loss of all body hair (alopecia universalis).

Alopecia areata is thought to be an autoimmune disease in which the immune system attacks the hair follicle, although the follicle is not destroyed since hair can re-grow. There also appears to be a hereditary component to the disease and a team lead by investigators at Columbia University Medical Center has now identified eight regions in the genome that are linked to the condition. The associated regions include some that have been linked to other autoimmune diseases including type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, celiac disease, and systemic sclerosis. Of particular interest for its potential role in the onset of disease is the ULBP (cytomegalovirus UL16-binding protein) gene cluster that has not previously been associated with autoimmune disease. Expression of ULBP3 proteins, which act as activating ligands for the NKG2D receptor on natural killer cells, is markedly upregulated in hair follicles affected by alopecia areata.

The study is published in the journal Nature.

Since drugs that target some of the pathways involved in alopecia areata have already been developed to treat other autoimmune diseases, the researchers hope that their discovery will lead quickly to treatments for hair loss caused by alopecia areata. The team are also developing a genetic test that should be able to help predict the likely course of the disease in a particular individual.

NSAIDs and Cancer – Another Piece of the Puzzle

Regular use of NSAIDS has been linked to reduced incidence of certain types of cancer but the underlying protective mechanisms are unclear. Some of the anticancer effects are believed to be mediated through inhibition of COX-2, but a study led by investigators at Sanford-Burnham Medical Research Institute has now identified another mechanism by which the sulindac sulfide (the NSAID metabolite of sulindac) inhibits tumour growth. The team found that sulindac sulfide induces apoptosis by binding to retinoid X receptor-α (RXRα), a member of the nuclear hormone receptor family which had been already been identified as a potential target for cancer therapy. In cancer cells, levels of RXRα are often reduced, at least in part because of proteolytic processing to a truncated form, tRXRα. As with other nuclear receptors, RXRα regulates transcription of target genes by binding to DNA response elements but accumulating evidence suggests that RXRα may also have extranuclear activity. Both RXRα and tRXRα can exist in the cytoplasm and the study showed that cytoplasmic tRXRα can activate the PI3K/AKT survival pathway by interaction with the p85a subunit of PI3K, leading to anchorage-independent cell growth in vitro, and tumour growth in animals. Sulindac sulfide was found to inhibit the tRXRα-mediated PI3K/AKT activation, suggesting that the compound could provide a useful lead for anti-cancer drugs targeting this pathway.

The use of NSAIDs to reduce the incidence of cancer has been limited by the risk of major cardiovascular events and the Sanford-Burnham have identified an analogue of sulindac sulfide, K-80003 which has improved affinity for RXRα but lacks significant COX-2 inhibitory activity. K-80003 inhibited the growth of cancer cells in vitro and in animals and would be expected to have reduced COX-2-associated side effects.

The study is published in the journal Cancer Cell.

Cutting off Lipid Supply Halts HCV

New research has shown that compounds that affect cellular energy status could also be used to treat hepatitis C virus (HCV) infections. Metformin, which is used to treat type II diabetes, and 5-amino-1-β-D-ribofuranosyl-1H-imidazole-4-carboxamide (AICAR), which has been shown to mimic the beneficial effects of exercise in mice, stimulate AMP-activated protein kinase (AMPK). AMPK is a key sensor of cellular energy status and regulates both lipid and glucose metabolism to maintain cellular energy balance and protect against metabolic stress. An increase in the AMP/ATP ratio initiates an AMPK-mediated switch from activities that consume ATP, such as lipid production, to activities that produce ATP, such as lipid and glucose oxidation.

Infection with viruses might be expected to activate AMPK because of the energy demands put on the cell by viral replication, but research led by scientists at the University of Leeds has shown instead that HCV switches off AMPK so that the cell continues to produce the lipids needed to provide new viral particles with a protective outer coat. When the team treated HCV-infected cells with metformin or AICAR, AMPK activity was restored and viral replication was inhibited.

The team plan to carry out a small scale clinical trial to investigate the effects of AMPK activators in HCV infection and hope that such drugs may provide much-needed new treatments for HCV.

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

Cholesterol – All in the Mind?

Cholesterol is essential for all animal life but high levels of cholesterol – when associated with low density lipoprotein (LDL) – are linked to an increased risk of atherosclerosis, heart disease and stroke. Circulating cholesterol can also be transported by high density lipoprotein (HDL); HDL is believed to be able to remove cholesterol from atheroma within arteries and cholesterol associated with HDL is considered to be beneficial for cardiovascular health. Cholesterol levels are determined by dietary intake, de novo synthesis and secretion by the liver: cholesterol absorption blockers and HMG-CoA reductase inhibitors (statins), which block cholesterol synthesis, are used clinically to reduce cholesterol levels.

A study led by researchers at the University of Cincinnati has now identified a new potential target for the control of cholesterol. The study, carried out in mice, found that circulation of cholesterol is regulated in the brain by the ‘hunger hormone’, ghrelin, which inhibits the melanocortin 4 receptor (MC4R) in the hypothalamus and is important for the regulation of food intake and energy expenditure. Increased levels of ghrelin were associated with increased levels of circulating HDL cholesterol, which the authors attribute to a reduction in the uptake of cholesterol by the liver. Genetically deleting or chemically blocking MCR4 in the CNS also led to increased levels of HDL cholesterol, suggesting that MCR4 is key to central control of cholesterol.

More studies are need to determine whether the effects observed in mice can be applied to humans but the finding that a neural circuit may be directly involved in the control of cholesterol metabolism by the liver could provide a target for new treatments to control cholesterol.

The study is published in the journal Nature Neuroscience.

Kinase Target for Toxoplasmosis, maybe Malaria

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.

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.