IBD: proteases offer new targets for drug discovery

Final Report Summary - IPODD (Inflammation bowe disease(IBD): proteases offer new targets for drug discovery)

IPODD was launched in 2008 with a EUR 3 million funding from the European Union (EU)'s Seventh Framework Program (FP7). The project put together the expertise of 13 research teams across 7 countries with the goal of pursuing new strategies for treating inflammatory bowel disease (IBD).

IBD affects about 1 in every 250 people in Europe; they are chronic disorders of the gastrointestinal tract that include two main sub-types, Crohn's disease (CD)and ulcerative colitis(UC).Both diseases involve symptoms ranging from abdominal discomfort and diarrhoea to severe abdominal pain, rectal bleeding, anaemia, and weight loss. Severe disease often results in surgery to remove damaged parts of the bowel. Removing all of the colon is an effective strategy to cure UC, but the effects of having a permanent stoma are stigmatising, especially in young people. CD is incurable. IBD is therefore a major cause of disability and morbidity in western countries, interfering with personal and professional life of tens of thousands of young, active people.

IPODD took a pragmatic approach to the study of IBD: the project focused on the final steps of the inflammatory cascade in the gut, namely the control of the molecules (proteases) that destroy the tissue blocking. These final steps may lead to new therapies to prevent severe disease.

Project Context and Objectives:

Contractors involved

IPODD teamed up 13 principal investigators in 7 countries during its 3-years duration.

List of contractors

- Academic Medical Centre, Amsterdam (Guy Boeckxstaens)
- Alimentary Health Limited, Cork (Jennifer Roper)
- Barts and the London School of Medicine and Dentistry, London (Thomas MacDonald,David Bulmer)
- Eberhard Karls Universität Tuebingen (Paul Enck)
- GlaxoSmithKline Research and Development Ltd, Stevenage
- Institute of Microbiology AS CR, Prague (Helena Tlaskalova)
- Technische Universität München, Munich (Michael Schemann, Dirk Haller)
- The University of Auckland, New Zealand (Lynnette Ferguson)
- University College Cork (Fergus Shanahan)
- University of Sheffield, (David Grundy)
- University of Southampton (Sylvia Pender)
- University Tor Vergata, Rome (Giovanni Monteleone)

Note: GlaxoSmithKline withdrew from the consortium in 2010 following an internal restructuring of the company.

The two main types of IBD, CD, and UC, have many features in common. In both disorders, patients suffer from chronic inflammation of the intestine and often experience periods of relapse (also known as 'flares') and remission, during which symptoms may be mild or absent. Some CD's patients may also suffer from the narrowing of the intestinal lumen (strictures) due to fibrosis, a consequence of chronic inflammation. In CD, inflammation can occur anywhere in the digestive tract but usually causes ulcers along the small and large intestine, whereas UC affects the colon and rectum.

The exact cause of IBD is still unknown. A widely accepted hypothesis is that microbes normally living in the intestine may trigger an aberrant immune response in genetically predisposed individuals, leading to chronic inflammation. In fact, 99 different gene variants are associated with IBD suggesting that development of disease in an individual is complex and there may not be a single cause.

Current treatment for IBD relies on immunosuppression. Corticosteroids, the mainstay of treatment, have unacceptable side effects with long terms use. More potent immunosuppressive drugs, such as azathioprine can provide long-term benefit in CD, but neither corticosteroids nor azathioprine alter the course of the disease, providing only symptomatic relief. More modern immune modulators, such as antibodies against tumor necrosis factor (TNF), are useful in patients with severe disease. However, many patients fail to respond to any therapies, eventually requiring surgery either because their disease resists treatment or because of complications such as strictures, perforations and abscesses.

Therefore, despite a significant progress in the management of IBD patients, there is clearly the need for newer therapeutic alternatives.

IPODD's work focused on matrix metalloproteases (MMPs), a family of enzymes that lie at the end of the inflammation cascade. These molecules do not trigger inflammation themselves, but are involved in its final steps. About two dozens MMPs are known in humans. Because of their ability to digest proteins (proteases is a general term to indicate 'protein-breaking enzymes'), MMPs are a sort of 'demolition squads' in the vanguard of inflammation. They dissolve proteins in the matrix (the space surrounding cells), clearing the way for inflammatory white blood cells. MMPs may also attack the structural proteins that hold the gut wall together, resulting in destruction of the intestinal tissue, and severe ulceration. Other proteases, like the TNF converting enzyme (TACE) are important players in inflammation: TACE is very important in the production of TNF, a cytokine with strong pro-inflammatory effects. However, proteases do not function in isolation. They are tightly controlled so that all proteases in the body also have inhibitors of their function. For MMPs, the inhibitors are the tissue inhibitors of metalloproteases (TIMPs). It is thought that tissue destruction occurs when the activity of MMPs overwhelms the ability of TIMPs to protect tissues.

Project results:

IPODD was the first consortium to focus specifically on MMPs and their multifaceted role in IBD. The multidisciplinary approach taken by the consortium was reflected in the wide range of expertise of its groups, with specialists in immunology, microbiology, probiotics, neuro-gastroenterology, genetics, drug development and science communication. The project contained nine work packages (WP) detailed in the project periodic reports and the respective deliverable reports submitted to the European Union (EU).

At the outset of the project:

- it was unclear which proteases were most relevant to IBD or their specific role in disease;
- little was known about the association between protease genes and IBD susceptibility;
- there was no available strategy specifically to inhibit MMP action in animals or man;
- there were little or no data about the role of MMPs and their inhibitors produced by the intestinal bacteria, and their relevance to IBD;
- the role of proteases in the perturbation of the enteric nervous system during inflammation was poorly understood.

Overall, the IPODD work clarified the role of several proteases in the pathogenesis of IBD, and produced data to suggest that they may be rejuvenated as therapeutic targets.

Identifying the role of MMPs and their inhibitors in IBDs

A core component of IPODD's work was to find which proteases are present in the gut in IBD and to study their specific role in the disease. Researchers compared the expression of proteases in normal gut and in those affected by IBD, and looked at the role of MMP gene variants in patients. These studies provided a clearer picture of the significance of proteases in chronic gut inflammation and identified some MMPs as possible targets for drugs that may inhibit their activity. The role of TACE, a major pro-inflammatory factor, was also an important field of investigation.

Key results include:
- DNA studies identify associations between variants in MMP genes and IBD in patients from New Zealand and the Netherlands;
- characterisation of several MMPs as targets for possible inhibition by TIMPs or drugs;
- expression profiles of MMPs in normal and inflamed gut showed MMP3 and MMP12 are particularly prominent in IBD, while TIMP3, a natural inhibitor is decreased;
- the role of MMPs and their inhibitors in gut fibrosis was examined;
- evidence for TIMP3 control of TACE and TNF-'production in the human and mouse intestine.

Linking MMPs to neuro-immune mechanisms

Several studies have shown a link between nerves, the brain and the immune system in chronic gut inflammation. Nerves and immune mast cells are closely associated in the human colon. Moreover, many enteric neurons express protease-activated receptors (PAR) that can be activated by MMPs. This led IPODD to address the question of whether proteases, including MMPs, are involved in nerve-gut interactions. This field is critical to understand why IBD patients have an increased sensitivity to pain, and can explain why stress and other events affecting the brain are often associated with relapses ('flares'). IPODD researchers found evidence that proteases are involved in neuro-immune responses, including visceral sensitivity and stress-reactivated colitis. Preliminary results suggest that inhibiting proteases could reduce the sensitivity of IBD patients to gut pain and dampen the effect of stress on relapses.

Key results include:

- proof that extracts from inflamed gut samples of IBD patients stimulate enteric neurons in vitro. This effect is blocked by protease inhibitors, proving their role in neuro-immune mechanisms;
- identification of PAR1 as the main receptor involved in protease-mediated neuro-immune signalling in the human gut;
- evidence that the expression of several MMPs is increased in stress-induced colitis and may be related to the elevated sensitivity of patients suffering from IBDs;
- identification of TIMP-3 as a possible important biomarker for stress-induced colitis.

(re) Discovering the role of bacteria in IBD

One cannot overstate the importance of the trillions of microorganisms that live in the human intestine. An accepted model for IBD is that gut bacteria trigger an abnormal immune response in genetically predisposed individuals, leading to inflammation. IPODD found that bacteria are also involved in the late stages of gut inflammation: in fact, some MMPs are produced by bacteria, and not by the host. The consortium found evidence that bacterial proteases contribute to the development of inflammation. The study of enteric bacteria may lead to identify new factors involved in IBDs and to discover new inhibitors that could be used to control inflammation.

Key results include:

- discovery of bacterial proteases involved in IBD by screening biobanks of enteric bacteria and samples from patients;
- proof that bacterial proteases may contribute to the pathogenesis of IBD, with profiles differing between CD and UC.

Developing strategies to inhibit MMP activity

A significant part of IPODD's work was aimed at testing methods that may block the effect of MMPs in the human intestine. By counteracting the action of proteases with specific drugs, researchers from the consortium managed to reduce inflammation in animal models of IBD. They also found that a probiotic mixture used in the management of IBS and UC (VSL#3) has protease-inhibiting properties, which may in part explain its effectiveness. More research in this field may help design better probiotic therapies for IBDs.

Key results include:

- significant reduction of colitis in mice treated with a selective inhibitor of MMP 3/12;
- reduction of inflammation with a PAR-2 antagonist in murine models of colitis;
- identification of a protease-inhibiting effect of VSL#3, a probiotic mixture used in IBD;
- identification of an individual lacking TACE. The patient does not have significant intestinal symptoms despite his impaired ability to produce an active TNF. This shows it may be possible to inhibit TACE in the gut without major side effects.

Potential impact:

During its three-years of activity, IPODD engaged in several initiatives to disseminate scientific knowledge and results across the project research teams involved and the lay public. Summer schools, consortium meetings and training schemes for young researchers ensured the internal transfer of know-how, while an international symposium and initiatives with patients' associations were key to publicise IPODD's work to a lay audience.

Key results include:

- summer schools attended by 15-25 young investigators (Tuebingen 2010, Rome 2011);
- three annual training fellowships granted to young researchers;
- three annual meetings (Prague 2009, Tuebingen 2010 and Rome 2011) and a two-day international public symposium (Rome 15 - 16 April 2011);
- an IPODD public summary about the consortium's work, mainly targeted to patients.

Translation and dissemination of the summary in 13 EU countries across a network provided by the European Federation of CD and UC Associations (EFCCA).

Impact and perspectives beyond IPODD

IPODD's work has greatly contributed to improve knowledge of the late stages of inflammation and the role of proteases in IBD. Results arising from the consortium, in particular, highlighted the importance of the balance between pro-inflammatory cytokines, MMPs and their inhibitors in the pathogenesis of IBD, and identified key players in the process. A next step will be to screen for compounds that inhibit proteases and test them as potential drugs for IBDs.

The work on bacterial proteases shed new light on the importance of enteric bacteria and will prompt researchers to mine these microorganisms for natural inhibitors of MMPs that may also be useful as drugs. One perspective is to engineer gut bacteria to produce protease inhibitors and use these strains as a probiotic therapy. Further research will address the role of proteases in IBD in detail, and determine whether we can harness enteric bacterial to counteract inflammation in patients.

Overall, IPODD has significantly advanced the IBD field, opening cutting-edge lines of research that in time may lead to new therapeutic approaches. Other fields, such as neuroimmunology and neuro-gastroenterology, could also benefit from the information and the skills accumulated by the consortium. This potential will hopefully be maintained and developed with future funding opportunities.

Final Report Summary - IPODD (Inflammation bowe disease(IBD): proteases offer new targets for drug discovery)

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