To begin, could you provide an introduction to your work and how you became interested in immune cell function?

I’m interested in understanding the mechanisms by which cells are regulated and the defects that occur in disease. Many human diseases are caused by inappropriate or chronic inflammation in which immune cells are activated, including colitis, asthma, arthritis, organ transplantation and Type 1 diabetes. However, many other diseases, such as Alzheimer’s disease, cancer and Type 2 diabetes are now also known to involve chronic inflammation. Thus, studying how to control inflammation will offer insight into how to intervene.

What is interleukin-10 (IL10) and what role does it play in cell function?

IL10 is a cytokine, a protein that is secreted by cells in response to specific stimuli and binds receptors on target cells to stimulate programmed responses. For instance, the presence of a pathogen stimulates an immune cell called the macrophage to produce pro-inflammatory cytokines such as tumour necrosis factor alpha (TNFα).

TNFα is essential for the host to defend itself against pathogens; it recruits and activates

immune cells to help eliminate the pathogen. However, an essential part of the defence mechanism is the orderly deactivation of the recruited immune cells once the pathogen is eliminated.

IL10 is a key anti-inflammatory cytokine produced during the later phase of the inflammatory response. It binds to IL10 receptors (IL10Rs) on immune cells and shuts down the activation cascade. The importance of IL10 in moderating the inflammatory response is shown by the sustained and pathological inflammation in humans (and mice) lacking IL10 or its receptor.

Could you outline the key objectives of your current work?

My first goal is to characterise the intracellular signal transduction pathways activated by IL10 binding to its receptor, which deactivate the cell. We have found that the SHIP1 inositol phosphatase signal transducer and activator of transcription 3 (STAT3) transcription factor regulated pathways are important but data suggest that other pathways exist. We want to find out the nature of these pathways and how they complement each other.

My second objective is to understand the structural changes occurring during the activation/deactivation cycle of the SHIP1 enzyme. SHIP1 activity becomes activated by the IL10R through binding to a SHIP1 agonist. We have isolated small molecule drugs that can activate SHIP1 and mimic the biological function of IL10. We are using biophysical techniques to determine how these agonists work.

Could you discuss the high-throughput SHIP assay you developed to screen for small molecule activators of SHIP activity?

The assay is completed in plate format using a recombinant SHIP1 enzyme (+/- test compounds) and a water soluble version of the SHIP1 substrate called inositol-1,3,4,5-tetrakis phosphate (IP4, the non-acyl

version of PIP3). SHIP1 dephosphorylates IP4 to release IP3 and inorganic phosphate, which can be detected with a dye that turns green when it binds inorganic phosphate. Compounds that activate SHIP1 activity therefore turn the well greener in colour.

How will your research lead to strategies to treat diseases such as ulcerative colitis?

The immune system in the gut must remain tolerant of food and commensal bacteria, but react against pathogenic bacteria. In colitis, the balance between anti- and pro-inflammatory reactions is skewed toward inflammation. Our SHIP1 agonists are able to inhibit inflammation and restore normal colon architecture in mouse models of colitis. A spin-off biotechnology company is currently testing SHIP1 agonists in human phase II clinical trials for the treatment of inflammatory diseases.

Are you engaged in any important collaborations?

We are working with other investigators to examine the potential therapeutic application of SHIP1 agonists in reversing tumour-induced immune suppression. Tumours produce factors that elevate the numbers of suppressive immune cells. We have found that SHIP1 agonists can in turn reduce the number of these suppressive cells. This might allow the immune system to help eliminate the tumour.

What will be the focus of your research in coming years?

We plan to examine the mechanism involved in IL10 deactivation of cell types other than macrophages. For example, IL10 is often produced by regulatory T cells to inhibit effector T cell function. We are also interested in developing small molecule agonists that can activate other inositol phosphatases such as phosphatase and tensin homolog (PTEN) and SHIP2, which could be used to treat diseases resulting from reduced function of these phosphatases.

Biochemist Dr Alice Mui is exploring what happens when the immune system is over activated. Here, she explains the ubiquity of these mechanisms, her fascination with a class of cell-signalling molecules, and the potential of her work to treat a plethora of diseases

Controlling inflammation

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DR ALICE M

UI

ALL THINGS NEED an ‘off switch’, and the immune system is no different. When this fails, and activated immune cells are not turned off when they have done their job, a host of inflammatory disorders, such as rheumatoid arthritis and diabetes, become manifest.

This is an intricate process involving a multitude of factors. When an immune cell called the macrophage is activated by a pathogen, it produces a suite of chemical factors that protect the body against the oncoming challenge. One set of these factors is called cytokines, proteins that recruit immune cells to the injury site. As the pathogen is eliminated, the macrophage begins to produce chemicals that ‘turn off’ the pro-inflammatory response and aid the crucial healing phase. One particularly important factor here is interleukin-10 (IL10), an anti-inflammatory cytokine that negatively regulates immune cell function, which is critical to ensuring inflammatory responses to do not lose control and cause disease.

Dr Alice Mui, Professor in the Departments of Surgery and Biochemistry, and Molecular Biology at the University of British Columbia (UBC), Canada, is fascinated by the mechanisms through which IL10 turns off immune cells, a process that goes awry in disease. In revealing this, she hopes to offer treatments that mimic IL10’s beneficial properties.

CYTOKINE PROGRESS

Mui has long been interested in how cytokines transduce signals to generate biological responses. As a graduate student she purified the then uncharacterised IL3 receptor and obtained its amino acid sequence, shedding new light on IL3’s role in cell proliferation. She moved on to IL10 as a senior research associate, revealing a new function for the molecule: inhibition of macrophage proliferation. Now applying a more translational approach to her work, Mui is focusing on developing therapeutic applications based on her findings.

Her team at UBC has conducted extensive research into the signalling pathways used by IL10 to suppress macrophage activation, making major contributions to the field. They defined a hitherto unknown activity for the cytokine, inhibition of macrophage proliferation, and showed that signal transducer and activator of transcription 3 (STAT3) is necessary for this function. But Mui also found, for the very first

time, a mechanism that is independent of transcription, through which IL10 suppresses macrophage activation. This pathway is responsible for IL10’s immediate inhibitory effects and is mediated by a signalling molecule called SHIP1.

DRUG DISCOVERY

Recognising SHIP1 as an ideal and completely novel target for anti-inflammatory drugs, Mui and her colleagues went on develop compounds that specifically activate SHIP1, thereby inhibiting inflammation.

This was a long process, made possible by the development of an innovative high throughput screen to find modulators of SHIP1 activity. Running this screen against a natural product library, Mui identified a small molecule agonist of SHIP1 – the first described small molecular activator of a negative signalling molecule. Collaborators in UBC’s chemistry department synthesised analogues of the natural product, which demonstrated an even greater activity than the original. Further tests showed these agonists to be potent against both inflammation and myeloma, representing an entirely new drug class. A UBC spin-off company called Aquinox now owns the license for these compounds and is further developing SHIP1 agonists, which are now in human phase II clinical trials for the treatment of inflammatory disease. Studies suggest they could also be used to treat cancers of the blood.

After showing the clear clinical benefits of the compounds, Mui’s lab proceeded to define the molecular mechanism by which they work. In doing so, they made another unprecedented discovery – that SHIP1 is allosterically regulated. All enzymes have an active site, which binds the substrate of the chemical reaction being catalysed. However, some enzymes, like SHIP1, have an additional site called an allosteric site. Here, effector molecules can bind to change the enzyme’s activity, often by modifying its conformation.

Not only did the team find SHIP1’s allosteric site, they also showed that the small molecule agonists bind to it to activate SHIP1. This was an important scientific discovery, but also allowed the researchers to perform molecular modelling to design better agonists, as well as antagonists (which inhibit activity) to fight diseases in which the immune system is underactive.

CLOSING KNOWLEDGE GAPS

More recently, the group found that IL10 stops the synthesis of a key pro-inflammatory cytokine – tumour necrosis factor alpha (TNFα). This takes place through a process that requires both SHIP1 and a phosphorylating enzyme called Mnk1. Yet, the precise molecular details of how SHIP1 is activated, and how this regulates signalling pathways, remain somewhat enigmatic.

However, the team has several hypotheses. The first of these states that signalling from the IL10 receptor (IL10R) involves activation of both SHIP1 and STAT3, and allosteric activation of SHIP1. To test this, Mui will characterise the interaction between these three molecules, and the structural changes taking place in SHIP1.

The team also believes IL10’s silencing of TNFα is mediated by Mnk1-regulation of a set of binding proteins called ARE. A series of studies will investigate whether ARE proteins are Mnk1 targets that do in fact regulate TNFα translation, and identify the proteins regulated.

Having shown the importance of SHIP1’s catalytic domains, Mui believes its other domains may also have important roles in its regulation of immune cell function. So, finally, she will conduct a number of studies in mice to pinpoint the contribution of these non-catalytic domains.

EXPANDING FOCUS

Through their concentrated research efforts, the Mui lab has made two major findings: IL10 uses an intracellular signalling protein called SHIP1 to deactivate immune cells, and SHIP1 is an allosterically regulated enzyme that can be activated by small molecules. This, in particular, was a hugely important finding, as it opened the doors to drug development.

Mui’s current work will advance on this, exploring how SHIP1’s activity is regulated by IL10 and allosteric activators; how the SHIP1 pathway interferes with the synthesis of inflammatory proteins; and how SHIP1 domains contribute to its immune regulation. Together, these studies will provide a better understanding of how IL10 and SHIP1 inhibit immune cell activation, which may lead to new and more effective therapeutics for inflammatory disease. These general principles could be expanded to other cell types as well, and even other enzymes, potential generating small molecules to treat a host of diseases.

Finding the ‘off switch’The activation of immune cells is crucial to fighting infection, but must be carefully regulated to avoid negative consequences. Researchers at the University of British Columbia are studying the mechanisms and molecules involved in the process, and developing drugs that exploit these properties

DR ALICE MUI

106 INTERNATIONAL INNOVATION

NEGATIVE REGULATION OF IMMUNE CELL FUNCTION

OBJECTIVES

To understand the mechanism by which interleukin-10 (IL10) and SHIP1 act to turn off immune cells such as macrophages.

PARTNERS

Dr Chris Ong, Department of Surgery, UBC

Dr Gerald Krystal, Department of Pathology and Laboratory Medicine, UBC

Dr Raymond Andersen, Department of Chemistry and Oceanography, UBC

FUNDING

Canadian Institutes of Health Research (CIHR)

CONTACT

Dr Alice Mui Assistant Professor

University of British Columbia Vancouver Coastal Health Research Institute Jack Bell Research Centre 2660 Oak Street Vancouver, British Columbia V6H 3Z6 Canada

E alice.mui@ubc.ca

www.biochem.ubc.ca/person/alice-mui

DR ALICE MUI’s laboratory studies the mechanism by which negative regulators like IL10 inhibit target cells in order to intervene therapeutically during deregulated immune responses. Her group has identified an agonist of the inositol phosphatase SHIP1, which mimics the anti-inflammatory action of IL10. Her laboratory confirmed that the agonist inhibits inflammation in animal models of septicemia and colitis; both diseases resulting from over-enthusiastic activation of the immune system. The agonist has now been licensed to a spin-off company for clinical development for the treatment of human inflammatory disease. The Mui lab is now characterising small molecule SHIP1 inhibitors which might be used to reverse pathogen-induced immunosuppression. Work is also continuing towards characterising other signalling pathways utilised by IL10 and other negative immune cell regulators.

SHIPping out

Mui summarises what makes SHIP1 such an excellent therapeutic target, and how its discovery as the first ever small molecule activator of a negative signalling molecule has impacted the world of drug discovery

Ideal therapeutic target

• SHIP1 is mainly expressed in immune and blood stem cells, so drugs targeting the molecule will have a minimal effect on the other cells of the body, and thus few side effects

• Because SHIP is an enzyme, drugs activating it will have a rapid response

• SHIP1 is not a target of any existing drugs, so activators should work well in combination with other drugs

Progress since identification

• SHIP1 agonists are already in human clinical trials for inflammatory disease

• Efforts are underway to identify activators of other phosphatases

• Allostery caused by conformational change is also being explored in the context of other molecules

INTELLIGENCE

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