Alpha-1 Antitrypsin Deficiency – research update

Now almost a year into a two-year project, Dr Bibek Gooptu, from the Institute for Structural and Molecular Biology (ISMB) at Birkbeck and UCL, and his team are researching new treatments for the condition Alpha-1 Antitrypsin Deficiency and helping to advance our understanding of the mechanisms behind the condition.

The protein Alpha-1 Antitrypsin is created in the liver and then carried in the blood to the lungs, where it protects lung tissue. In people with Alpha-1 Antitrypsin Deficiency, a mutant gene within the liver cells causes the Antitrypsin protein to adopt a different structure, and individual protein molecules link together in chains, known as polymers. The liver is not able to secrete these polymers and so they remain stuck inside liver cells, damaging them. It also means that not enough Antitrypsin reaches the lungs from the bloodstream. People with Antitrypsin Deficiency are likely to suffer both from liver disease (e.g. cirrhosis or cancer of the liver) and, if they are smokers, they are likely to develop emphysema at a much younger age than someone without Antitrypsin Deficiency – probably in their 30s or 40s.

Promising compounds

Dr Gooptu and the project team (Gooptu group and collaborators at Birkbeck, UCL and Cambridge) have been studying compounds which will bind to the Antitrypsin protein in such a way that it is prevented from forming polymers. Because they have previously solved the molecular structure of the Antitrypsin in atomic detail they have identified three possible drug binding sites to target with new treatments. They have confirmed that small molecules (drug building blocks) predicted to target these sites in computer simulations bind the protein when tested experimentally in solution. The team are currently finalising computer simulations identifying a set of 750 compounds with similar binding characteristics to these ‘hits’. They will use these to test run the technology they are developing to assess the effects of many molecules at a time, not just on the protein in a test tube (which they do using a technique called mass spectrometry) but also at the site where the disease starts: in cells.

Example of a compound (cyan) successfully binding one of the target sites (surface features shown in grey)  in Alpha-1 Antitrypsin (molecular structure shown as coloured ribbons) in computer simulation.

Example of a compound (cyan) successfully binding one of the target sites (surface features shown in grey) in Alpha-1 Antitrypsin (molecular structure shown as coloured ribbons) in computer simulation.

Testing in mammalian cells

Although a compound may bind to a protein in a solution, to be a successful drug the compound has to be able to enter the cell effectively. Once inside it must bind effectively in conditions that may be quite different. To see whether the compounds which bound successfully in solution can bind in the same way in cells like liver cells the team are testing them in cells known as CHO cells.  These are simple mammalian cells that grow well in a nutrient broth (cell culture) and behave like liver cells in terms of how they deal with normal Antitrypsin (secreting it efficiently) and disease mutant forms (accumulating polymers within the cell). It is already possible to check these cells for beneficial effects of drug-like compounds by checking whether the amount of secreted Antitrypsin goes up and/or the polymer levels go down. However at early stages of drug development the effects of the compounds may be more subtle, so Dr Gooptu and his post-doctoral researcher, Dr Nyon, are working with the Thalassinos group at UCL to identify more sensitive markers. For this they are working out the molecular signatures of health and disease in the part of the cell where proteins are prepared for secretion and where Antitrypsin polymers form and get stuck. These signatures are the levels of different proteins found within this area, known as the endoplasmic reticulum or ER for short. Not only is this useful for the current project, the signatures may themselves provide clues for other treatment options in the future.

Other proteins can aggravate or lessen disease

As well as the Antitrypsin protein there are hundreds of other proteins present in the ER. The amounts in which these proteins are present change depending on whether the cell is healthy or unhealthy. Some drop to undetectable levels while some new proteins are only found in one or other situation. Dr Gooptu compares this to looking at a street from an aerial view. In one view there are people in the street, with music and bunting. This is a street party and the equivalent to the ER of a healthy cell. In another view of the same street there might again be people in the street, but this time you also observe flames, fire engines and hoses. This is an emergency situation and the equivalent of the ER in an unhealthy cell. However, it is necessary to establish whether the people (proteins) in the second scene are causing the disease (arsonists) or fighting it (fire fighters). This will allow comparison of desirable with harmful responses to promising compounds and identify other proteins that might themselves be useful targets for future drug treatments.

Understanding how compounds are binding

Once they have identified the best compounds from the original set of 750 that bind Antitrypsin both in free solution and within cells, a technique known as Nuclear Magnetic Resonance (NMR) spectroscopy will be used to look very closely at the binding process and discover exactly how it takes place.

Binding of the Antitrypsin protein (grey ribbon representation) to a prototype drug molecule can be followed by NMR looking at changes seen when the interaction occurs around the structure at many individual points (spheres). In this case the colour coding shows many areas that change a lot (blue), whilst a few areas are stable (white)

 

Binding of the Antitrypsin protein (grey ribbon representation) to a prototype drug molecule can be followed by NMR looking at changes seen when the interaction occurs around the structure at many individual points (spheres). In this case the colour coding shows many areas that change a lot (blue), whilst a few areas are stable (white)

 

 

Using this technique the team can identify the characteristics of the protein:compound interaction. Armed with this information they can return to a library of thousands of compounds and identify further potential binders for testing and develop compounds which will bind in the most effective way.

Improving understanding of Alpha-1 Antitrypsin deficiency and the disease mechanisms

The approach taken by Dr Gooptu has developed existing methods and combined them in a new way. Usually drug companies will blind-test thousands of compounds in solution. Using the computational modelling before the in-solution testing meant that the team could identify a smaller number of compounds which were more likely to give positive results. The more detailed NMR spectroscopy studies are then targeted on a small number of compounds that show the most promise both in solution and in cells. This approach also has the additional advantage that while identifying compounds that bind Antitrypsin it also reveals more about how the genetic mutation causes disease in terms of harmful changes in proteins and cell responses.

Next steps

Having carefully developed the individual computational, mass spectrometry, cell biology and NMR techniques over the last year, in the next 12 months the project team will put them together and see how well they work in a pipeline for assessing the 750 test compounds. If this works well the pipeline could then be boosted to screen far greater numbers of compounds in future. However above the molecular scale, the project has already paved the way for bigger drug discovery projects in Alpha-1 Antitrypsin deficiency. Through his work, and the forum of research meetings convened by the US patient charity that funds the project (the Alpha-1 Foundation), Dr Gooptu has established contact with other groups working on a range of other approaches to identify new treatments. He is now collaborating with these groups and the Foundation to develop larger screens in which hit molecules can be rapidly identified from cell screening. The hits will then be studied in parallel by his group in Birkbeck and US research groups with complementary expertise to establish how they work and so how they can be further improved as efficiently as possible.

Dr Bibek Gooptu has been working on Antitrypsin Deficiency at Birkbeck since 2006. He is also a practising Consultant in Respiratory Medicine.  His research is supported by the Alpha-1 Foundation, the Medical Research Council and the Wellcome Trust.

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Research into Alpha-1 Antitrypsin Deficiency

Dr Bibek Gooptu, from the Institute for Structural and Molecular Biology (ISMB) at Birkbeck and UCL, has been awarded a grant by the Alpha-1 Foundation for research into new treatments for the condition Alpha-1 Antitrypsin Deficiency.

Alpha-1 Antitrypsin Deficiency
Alpha-1 Antitrypsin is a protein which is created in the liver and then carried in the blood to the lungs, where it protects lung tissue. In people with Alpha-1 Antitrypsin Deficiency, a mutant gene within the liver cells causes the Alpha-1 Antitrypsin protein to adopt a different structure, which causes individual protein molecules to link together in chains, known as polymers. The liver is not able to secrete these polymers and so they remain stuck inside liver cells, damaging them. It also means that not enough Alpha-1 Antitrypsin reaches the lungs from the blood stream. People with Alpha-1 Antitrypsin Deficiency are likely to suffer both from liver disease (e.g. cirrhosis or cancer of the liver) and, if they are smokers, they are likely to develop emphysema at a much younger age than someone without Alpha-1 Antitrypsin Deficiency – probably in their 30s or 40s.

One in 27 people in Northern Europe carry one copy of the mutant gene for Alpha-1 Antitrypsin Deficiency, but for the disease to manifest, two copies are required.

Understanding Alpha-1 Antitrypsin Deficiency
Dr Gooptu’s research aims to understand why the mutation in the gene causes the structure of Alpha-1 Antitrypsin to change from the healthy structure. The ultimate objective is to find a way of stopping the Alpha-1 Antitrypsin forming polymer chains, so that the liver can secrete it and it can carry out its correct function – protecting lung tissue.

Finding new treatments
Typically drug development is either done by blindly testing large numbers of existing compounds to see if any show promise (“high throughput screening”), or else by very carefully designing a molecule so it works very well in theory, and then testing them out (“rational drug design”). The approaches have opposite strengths and weaknesses but at the ISMB Dr Gooptu’s team will combine them to get the best of both worlds. In the first stage Dr Gooptu’s team will take around 750 promising compounds and direct them at Alpha-1 Antitrypsin. Using a method known as mass spectrometry, he and his colleagues will identify those compounds which successfully bind the protein.

In the second stage, those compounds which interact most successfully with Alpha-1 Antitrypsin will be tested within cells, to see whether they cause a decrease in the formation of polymer chains, cell damage and/or an increase in Alpha-1 Antitrypsin secretion.

In the third stage, using Nuclear Magnetic Resonance (NMR) techniques, the team will look closely at the structure of the protein and the compounds in solution, to try to understand which binding sites they are using and how the binding is taking place. This information will enable the team to go back to much larger libraries of compounds and test new ones which are related to those which were most successful in cells, but should work even better. The team will therefore tailor the compounds based not just on ‘trial and error’ but instead combining practical information with knowledge of how the binding process is taking place.

By combining high throughput screening with studies in cells and NMR techniques, Dr Gooptu and his team hope to develop new drug treatments for Alpha-1 Antitrypsin Deficiency.

This research is funded by the Alpha-1 Foundation. It will start in July 2012 and run for two years.

Dr Bibek Gooptu has been working on Alpha-1 Deficiency at Birkbeck since 2006. He is also a practising Registrar in Respiratory Medicine.

10 July 2012
An additonal message from Dr Gooptu:

Since this post went up a number of people have contacted us, both on- and off-thread, expressing interest in the work, including offers to assist as subjects in the research.  In some cases people are getting in touch because of personal/family experience of this relatively under-reported condition.  The feedback is very much appreciated.  It is wonderful to have confirmation that our research is not an abstract intellectual bubble occurring inside ivory towers, but is of wider interest to the community as a whole.  We hope to post updates, via this blog, every 6 months or so and we can email anyone who wishes to be notified as and when these go up.

It should be emphasised that the research underway at Birkbeck and UCL is not at a stage where trials in humans are planned within the next few years.  On the other hand our group’s work sits within a strong network of researchers involved in a range of studies of alpha1-antitrypsin deficiency both within the ISMB; and elsewhere (in London, the wider UK and beyond).  Sometimes samples (e.g. blood samples) from individuals can be very useful in such studies.  Also, from time to time, ethically approved drug trials are conducted in alpha1-antitrypsin deficiency.

I can be contacted directly by email (mailto://b.gooptu@mail.cryst.bbk.ac.uk) about any of these issues, and will do my best to respond in a timely manner. For individuals who know they have alpha1-antitrypsin deficiency because they have 2 copies of the deficiency gene, and are interested in helping with ongoing research, an international registry has been set up based in Europe and the US.  The situation with carriers (one normal + one deficiency gene) is more complex.  Studies so far indicate the increased risk of disease in these individuals is very small indeed.

Bibek Gooptu

Useful resources that can be accessed online include*:

UK patients’ groups *
http://www.alpha1.org.uk/
http://www.alpha1awareness.org.uk/welcome.htm

US patient resources *
http://alpha-1foundation.org/
http://www.alpha1.org/

BBC info page
http://www.bbc.co.uk/health/physical_health/conditions/alpha1.shtml

British Lung Foundation
http://www.blf.org.uk/Home

American Thoracic Society/European Respiratory Society Guidelines on Diagnosis and Treatment of Alpha1-Antitrypsin Deficiency * http://www.thoracic.org/statements/resources/respiratory-disease-adults/alpha1.pdf

* Current treatment of alpha1-antitrypsin deficiency is slightly different in the UK to that practised in Europe and the US

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