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Targeting Orphan Receptors for Multiple Sclerosis

ROR-alpha ligand binding domain

Crystal structure of the ligand-binding domain of RORα complexed with cholesterol sulfate. PDB ID=1S0X

Scientists at the Scripps Research Institute have reported on compounds that are able to suppress severity and disease progression in animal models of multiple sclerosis. The compounds, exemplified by SR1001, act by selectively suppressing a subset of T-helper cells characterised by their production of interleukin-17 (TH17 cells). TH17 cells have been implicated in a variety of autoimmune diseases including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and systemic lupus erythematosus.

SR1001 selectively binds to two orphan nuclear receptors: retinoic acid receptor-related orphan receptors α and γt (RORα and RORγt). These receptors have indispensible roles in the development and function of TH17 cells, providing a mechanism for modulating one component of the immune system without general immunosuppression. The team reports that SR1001 induces a conformational change in the receptors that results in their reduced affinity for co-activators and increased affinity for co-repressors. The net result is inhibition of the receptors’ transcriptional activity.

SR1001 blocked the development of murine TH17 cells and inhibited cytokine production by differentiated murine and human TH17 cells. Although a drug is some way off, the team suggests that the results demonstrate the feasibility of targeting TH17 cells and the potential of such an approach for the treatment of autoimmune diseases.

The study is published in Nature.


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.


Genetic Basis for Alopecia Areata

Alopecia Areata

Alopecia areata

Image: Wikimedia Commons

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.


Beating Off the Attack in RA

seagulls

Image: Flickr - Jsome1

Although the exact causes of autoimmune diseases such as rheumatoid arthritis are unknown, tissue damage is mediated, at least in part, by the (inappropriate) action of effector T cells. Regulatory T cells are a specialised population of T cells which play a role in ‘self-tolerance’ by suppressing activation of other immune cells: genetic deficiency in regulatory T cells causes severe autoimmune disease in both mice and humans.

Researchers at NYU Langone Medical Center examined blood samples from 25 patients with rheumatoid arthritis and found that regulatory T cell function was inversely correlated with disease severity. The team have now discovered a way to enhance the activity of regulatory T cells which may eventually lead to new treatments for autoimmune diseases such as rheumatoid arthritis or inflammatory diseases such as Crohn’s disease.

In effector T cells, the calcium-independent protein kinase C, PKC-θ, localises at the site of contact between the T cell and the antigen presenting cell, placing it in the proximity of other enzymes that mediate T cell activation. When regulatory T cells become activated, however, the team found that PKC-θ is localised away from the point of cell-cell contact. In cell culture experiments, PKC-θ inhibitors were shown to boost the activity of regulatory T cells around 5-fold. One of the inhibitors was also shown to be protective in a mouse model of Crohn’s disease.

The study is published in Science Express and the PKC-θ inhibitors were provided by Boehringer-Ingelheim Pharmaceuticals, Inc.


Narcolepsy Established as Autoimmune Disorder

narcolepsy

Image: Flickr - Right About Me

Although the disorder is not very well known, narcolepsy is thought to affect 1 in 2000 individuals and this figure may be higher as a consequence of under-reporting and under-diagnosis. The most common symptom is excessive daytime sleepiness (EDS), which may be accompanied by sudden loss of muscular control (cataplexy) triggered by strong emotions. Narcoleptics may also experience sleep paralysis (short periods of paralysis when waking or falling asleep), hypnagogic or hypnopompic hallucinations (vivid images or sounds, respectively, when waking or falling asleep) or automatic behaviour (when routine activities are continued during a sleep episode).

For the last ten years it has been known that narcoleptics have a deficiency in hypocretin (orexin), a neurotransmitter involved in control of sleep/wakefulness. In parallel with the neurotransmitter deficiency there is a massive loss of hypothalamic neurons that produce hypocretin and it has been hypothesised that this results from an autoimmune response.

Swiss scientists have now identified autoantibodies to Tribbles homolog 2 (Trib2), an autoantigen previously identified in autoimmune uveitis, in narcolepsy patients. The team developed a transgenic mouse model to identify peptides enriched within hypocretin-producing neurons that could serve as potential autoimmune targets. Having identified enrichment of Trib2 in the mouse hypocretin neurons, the team went on to analyse sera from narcoleptics. Narcolepsy patients with cataplexy had higher Trib2-specific antibody titres compared with either normal controls or patients with other inflammatory neurological disorders. Trib2-specific antibody titres were highest early after narcolepsy onset, sharply decreased within 2–3 years, and then stabilized at levels substantially higher than that of controls for up to 30 years. Additionally, high Trib2-specific antibody titres correlated with the severity of cataplexy.

The study, published in the Journal of Clinical Investigation, provides the first evidence that narcolepsy is an autoimmune disorder.


PPAR-δ Agonists Could Help Lupus Sufferers

The name ‘lupus’ may have arisen because skin lesions resemble the bite of a wolf.  Image: Wikimedia - Natonal Park Service

The name ‘lupus’ may have arisen because skin lesions resemble the bite of a wolf.

Image: Wikimedia - Natonal Park Service

Systemic lupus erythematosus (SLE or lupus) is a chronic autoimmune disease that can affect any part of the body. Lupus cannot be cured, although symptoms can be managed and the risk of organ damage minimised by treatment with immunosuppressants, NSAIDS and corticosteroids. Lupus is a complex disease but, although the cause is unknown, a number of genetic susceptibilities and environmental triggers have been proposed.

Deficient phagocytic clearance of apoptotic cells by macrophages is one pathway that has been suggested to contribute to the pathogenesis of lupus. In healthy individuals, macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses to apoptotic debris. Scientists at Stanford University School of Medicine have now shown that the nuclear receptor, peroxisome proliferator-activated receptor-δ (PPAR-δ) plays a pivotal role in orchestrating phagocytosis. Genetically engineered mice lacking PPAR-δ showed decreased expression of opsonins, resulting in impaired clearance of apoptotic cells and reduced levels of anti-inflammatory cytokines. Both global and macrophage-specific Ppard-/- mice showed increased production of autoantibodies and were predisposed to autoimmune kidney disease, a condition resembling one of the manifestations of human lupus. PPAR-δ agonists are being investigated for the treatment of metabolic disorders such as hyperlipidemia, diabetes and obesity and the authors suggest that such compounds could also potentially benefit lupus sufferers.

The study was published online on October 18th in the journal Nature Medicine.


Homing in on Crohn’s Disease

colectomy sectionCrohn’s disease is an inflammatory disease of the gastrointestinal tract that causes abdominal pain, diarrhea and vomiting. First described by Burrill Bernard Crohn and co-workers in 1932, the disease is believed to be an autoimmune disorder but the precise causes are unknown. Treatment options focus on management of acute symptoms and maintenance of remission, since no known cure is available.

Crohn himself was convinced that the disease was caused by Mycobacterium paratuberculosis (MAP), the same pathogen responsible for the related Johne’s disease in cattle. Whilst his research was unable to establish the involvement of MAP, the theory has received more attention in recent years.

New research from McGill University Health Centre (MUHC), Quebec, has established a link between the human NOD2 gene and mycobacteria. Mutations in NOD2 have been observed in approximately 25% of Crohn’s disease patients, but the nature of the effect of these mutations has not been understood. Normally, NOD2 codes for a receptor that recognises invading bacteria, triggering an immune response. The MUHC study demonstrates that the NOD2 receptor preferentially recognises a peptide, N-glycolylated peptidoglycan-derived muramyl dipeptide (MDP), which is only found in mycobacteria. When mycobacteria invade the human body, they cause an immediate and very strong immune response via the NOD2 receptor. This new discovery, published in the Journal of Experimental Medicine, associates the predisposition for Crohn’s disease with both the NOD2 mutation and the presence of mycobacteria, but researchers must still determine the precise combination of these factors to understand how the disease develops.

In a separate study, researchers from Case Western Reserve University School of Medicine have identified a novel link between ITCH, a gene known to regulate inflammation in the body and NOD2. ITCH,which encodes an E3 ubiquitin ligase, can cause a variety of inflammatory diseases when malfunctioning. The team at Case Western found that ITCH also influences NOD2-induced inflammation. These findings, to be published in the August 11th issue of Current Biology, suggest a common pathophysiology exists between multiple inflammatory diseases. The unexpected finding of the interaction between these genes offers the possibility of new drug targets for intervention in Crohn’s disease.


Endolysosomal TLRs and Lupus

Lupus erythmatosusLupus erythematosus is a chronic autoimmune disease that, worldwide, is more common than leukaemia, multiple sclerosis, and muscular dystrophy. There are three recognised forms of lupus: discoid (cutaneous) lupus which affects the skin, systemic lupus (SLE) which attacks multiple organ systems, and drug-induced lupus which generally resolves once the drug is discontinued. The symptoms of lupus, which can flare and subside, vary from patient to patient but include severe fatigue, joint pain, muscle aches, anaemia, and general malaise. Approximately 10% of discoid lupus cases develop into systemic lupus which can result in the destruction of vital organs.

The causes of lupus are poorly understood, but researchers at the Scripps institute have now shown that specific Toll-like receptors (TLRs) play a key role in the development of this disease. One of the principal diagnostic indicators of lupus is a high level of self-recognising antinuclear antibodies. Antinuclear antibodies normally form part of the immune response and are produced when bacteria or viruses are degraded in the endolysosome. TLRs inside this compartment specifically detect viral RNA and viral and bacterial DNA and stimulate immune cells to produce antibodies against these molecules.

Writing in the Proceedings of the National Academy of Sciences, the Scripps team have shown that three TLRs in the endolysosome are necessary for the generation of antinuclear antibodies in mice. Previous studies had pointed to endolysosomal TLRs – TLR3, TLR7, TLR8 (in humans but not mice) and TLR9 – as important for the production of antinuclear antibodies but, in mouse models of lupus, knocking out only TLR 7 or TLR9 had not dramatically reduced symptoms. The Scripps team wanted to eliminate all three TLRs in mice and achieved this by knocking out a transmembrane endoplasmic reticulum protein, UNC-93B, which is necessary for correct functioning of the endolysosomal TLRs. Knocking out UNC-93B in strains of laboratory mice that spontaneously develop many of the same symptoms as human lupus sufferers produced animals with fewer antinuclear antibodies and fewer and less severe symptoms of lupus. Even lipid A stimulation of TLR4 – which is known to promote production of autoantibodies – did not produce symptoms of lupus in the knockout animals. Nucleic acid-sensing TLRs may thus provide a critical pathway in the development of systemic autoimmunity by reducing tolerance to nucleic acid-containing antigens.


New Trigger for RA

rheumatoid arhtritis handRheumatoid arthritis (RA) is a chronic, typically progressive, autoimmune disease that primarily affects the joints although it can damage other tissues, including heart, lung, and eyes. There is currently no cure for RA and the goal of treatment is reduce joint pain and inflammation, maximise function, and minimise joint destruction and deformity. Newer biological treatments targeting components of the immune system in combination with disease-modifying antirheumatic drugs (DMARDs) are effective in preventing joint damage in some patients, but not all sufferers respond to these treatments and some may relapse despite treatment. The cause of RA remains unknown but both genetic and environmental factors are suspected to play a role.

TLR4 crystal structureResearchers at Imperial College have now identified a new immune trigger that may contribute to the pathology of RA. Tenascin-C is an extracellular matrix glycoprotein specifically expressed at areas of inflammation and tissue damage in inflamed rheumatoid joints. The team found that injection of tenascin-C into the joint cavity in mice caused severe joint inflammation and damage and, in a separate experiment, that mice lacking tenascin-C were protected from erosive arthritis. In cultures of cells from rheumatoid arthritis patients, tenascin-C induced synthesis of pro-inflammatory cytokines via activation of Toll-like receptor 4 (TLR4). TLR4 is one of a family of receptors that play a key role in pathogen recognition and activation of innate immunity. Stimulation of TLR4 is known to activate macrophages leading to release of TNF-α, one of the targets of existing biological agents used to treat RA patients. Previous studies had shown that mice lacking TLR4 do not show chronic joint inflammation, and blocking the interaction between tenascin-C and TLR4 may provide a new way to combat RA.

The study is published in the journal Nature Medicine.


Selective Inhibition of the Immunoproteasome

Proteasomes are abundant and ubiquitous multi-protein complexes capable of degrading almost any protein into oligopeptides. The 20S proteasome is composed of 14 subunits (seven α and seven β) arranged in four rings to form a barrel, the ends of which can open to accept substrates. Catalytic activity is confined to three of the β-subunits, all of which are N-terminal threonine proteases. The addition of 19S regulatory complexes at the ends of the 20S multimer creates the 26S proteasome and confers the ability to process ubiquitylated substrates in an ATP-dependent manner.

Yeast 20S proteasome structureProteasomes chomp their way through a huge number of proteins including defective ribosomal products that arise from imperfections in the conversion of genetic information into proteins. The oligopeptides that result are then rapidly processed to constituent amino acids by endo- and amino-peptidases. Some oligopeptides, however, escape further processing and are presented on the cell surface by MHC Class I molecules. This provides a mechanism for the immune system to monitor the gene products that a particular cell is processing.

A variant of the proteasome, the immunoproteasome, is constitutively expressed in immune tissues and can be induced in other cell types in response to cytokines such as γ-interferon and TNF-α. The immunoproteasome differs from the proteasome at the level of the catalytic β-subunits and generates alternate peptides that modify the antigens presented by MHC class I.

The proteasome has been validated as a target in oncology and bortezomib was the first proteasome inhibitor approved in the US (2003). Until now, however, utility of proteasome inhibitors in auto-immune diseases has been hampered by the higher doses of conventional inhibitors required and the consequently smaller therapeutic window. This situation has potentially changed with results from Proteolix using their immunoproteasome-selective inhibitor, PR-957.

PR-957 is a peptide epoxy-ketone that selectively inhibits the LMP7 catalytic subunit of the immunoproteasome. Inhibition of this subunit modulates immune cell signalling and blocks production of cytokines associated with autoimmune inflammation, without affecting proteasome function in non-immune cells. In mouse models of rheumatoid arthritis, PR-957 treatment reversed signs of disease and reduced cellular infiltration, cytokine production and autoantibody levels. The study is published in the journal Nature Medicine.

Proteolix plans to file an Investigational New Drug Application for PR-957 in mid-2010.