Alpha-1-antitrypsin (AT) deficiency and the serpinopathies: pathobiology and new therapeutic strategies

Lead Research Organisation: University College London
Department Name: Medicine

Abstract

Antitrypsin is found at high concentrations in the bloodstream, where its main role is to protect the lungs against tissue damage from inflammation. Liver cells (hepatocytes) normally release individual molecules of antitrypsin into the circulation. Antitrypsin deficiency results when an individual inherits two genes with small changes in the antitrypsin protein. The Z variant, found in about 4% of people of North European decent, is the most common cause of severe antitrypsin deficiency. The Z variant causes antitrypsin to form long chains of linked molecules (called "polymers") that are trapped inside liver cells. The build-up of polymers damages the cell and increases the chance of developing liver cirrhosis and liver cancer. The reduced amount of properly formed protein in the circulation means the lungs are not as well protected against inflammation and so individuals develop emphysema. We have shown that a similar process occurs in mutants of other members of the protein family to which antitrypsin belongs. These include polymerisation of mutants of neuroserpin in the brain to cause dementia. We have grouped all the conditions together as a single class of disease that we have called 'the serpinopathies'. The application builds on 25 years of work by our group and has 5 interlinking projects within a programme of work. We propose to:

(i) define the structure of the pathological polymer using biophysical analysis and cryo-electron microscopy. We will define the structure of the pathological polymer isolated from human tissues and use this information to develop strategies to block the abnormal protein-protein linkage that underlies antitrypsin deficiency and the serpinopathies.

(ii) establish the cellular response to intracellular serpin polymers. We will use the imaging of cells that express polymers to define the effect of polymer formation on cell function. We will also establish whether cell dysfunction can be reversed by small molecules that block polymer formation and so determine whether it is the polymers themselves that cause cell dysfunction and disease.

(iii) define disease mechanism in a multicellular context and identify new therapeutic targets using a worm (C. elegans) model of antitrypsin deficiency. We have developed a novel worm (C. elegans) model in which Z antitrypsin is retained as polymers in association with impaired motility, thin body structure and delayed development. We will assess the effect of small molecules and polymer blocking antibodies on intracellular polymerisation and the motility of the worm, dissect the mechanism by which intercellular antitrypsin polymers signal toxicity within the cell and undertake a chemical screen for novel agents that can reduce the toxicity of polymers in the worm. These will be used as the basis to design new therapies for use in man.

(iv) define the dynamics of antitrypsin polymers in vivo and their utility as a biomarker of disease. We will study individuals with antitrypsin deficiency undergoing liver and lung transplantation to evaluate the changes in antitrypsin polymers within the lung and circulation. We will also evaluate circulating polymers as the first biomarker that is specific for predicting and diagnosising antitrypsin deficiency related liver disease.

(v) develop a diagnostic technology for imaging Z antitrypsin polymers in vivo. We will use cell penetrating monoclonal antibodies or small molecules that are specific for polymers to image intracellular polymers in mouse models of disease. Our longer term aim is to develop this for use in man so we can assess whether there is a correlation between polymer burden and liver disease and if this new imaging test will be useful to accelerate drug development in man.

Taken together this work will increase our understanding of mechanism of antitrypsin deficiency and the serpinopathies and allow the development of new approaches to treatment.

Technical Summary

We described the polymerisation of mutants that underlie antitrypsin deficiency and developed new paradigms for the liver and lung disease associated with this condition. We have also described the polymerisation of mutants of antichymotrypsin that have been associated with COPD and mutants of neuroserpin that cause a novel dementia that we called familial encephalopathy with neuroserpin inclusion bodies (FENIB). We grouped these diseases with others that result from polymerisation of mutants of the serpins as the serpinopathies. We have used biochemistry, biophysical analysis, crystallography, NMR, cell and induced pluripotent stem cells, monoclonal antibodies and Drosophila models of disease to dissect the structural basis of polymer formation and the cellular consequences. We are in late lead optimisation stage of developing small molecules that block polymerisation with GlaxoSmithKline. However it is critical that we understand the pathobiology of antitrypsin deficiency and serpinopathies in more detail in order to evaluate a broad range of therapeutic strategies with the aim of curing the disease. The aims of this programme are to: (i) define the structure of the pathological polymer using biophysical analysis and cryo-electron microscopy, (ii) establish the cellular response to intracellular serpin polymers, (iii) define disease mechanism in a multicellular context and identify new therapeutic targets using a C. elegans model of antitrypsin deficiency, (iv) define the dynamics of antitrypsin polymers in vivo and their utility as a biomarker of disease, (v) develop a diagnostic technology for imaging Z antitrypsin polymers in vivo. The work is enabled by the facilities at UCL and the shared lab in which the applicants work. Indeed the preliminary data have been generated following the move of Lomas and Sattelle to UCL. The findings from this programme are likely to be applicable to a broad range of other protein conformational diseases.

Planned Impact

There are many potential impacts of our work:

(i) Our work has led to one of only 8 'Discovery Partnership with Academia' (DPAc) agreements with GlaxoSmithKline to develop small molecules to treat antitrypsin deficiency. This has progressed to the late lead optimisation stage of drug discovery. There is still a long way to go and the proposed programme of work will provide enabling information for the small molecule campaign. However if it is successful then we will develop the first therapy for antitrypsin deficiency that aims to block intracellular polymerisation and increase the secretion of active protein. Moreover the work will provide a paradigm to target other intra-endoplasmic reticulum protein folding diseases. Our work has led to a BBSRC/GSK CASE Studentship to Sarah Faull and an EPSRC/GSK CASE Studentship to Alistair Jagger.

(ii) We have used antitrypsin deficiency as a test system to develop human induced pluripotent stem cell (hIPSCs) models of inherited metabolic liver disorders (that also includes glycogen storage disease Type 1a, familial hypercholesterolaemia, hereditary tyrosinaemia and Crigler Najjar syndrome). This has been used to spin out a company from the University of Cambridge (http://www.definigen.com) by Ludovic Vallier and one of our former PhD students (Tamir Rashid). One of my post-doctoral fellows (Adriana Ordóñez) worked for the company to generate cell lines for commercialisation.

(iii) The novel reagents generated by our programme (eg C. elegans and cell models) will be made available to the academic community and monoclonal antibodies will be commercialised as we have done for the 2C1 monoclonal antibody with Hycult Biotech (http://www.hycultbiotech.com/hm2289).

(iv) Our 2C1 monoclonal antibody detects circulating polymers of antitrypsin. We have shown that Individuals with a wide range of antitrypsin deficiency genotypes have circulating polymers (Eur. Resp. J., 2014;43:1501-1504). We are exploring with Grifols whetr the 2C1 monoclonal antibody can be used as a 'point of care' screening test to identify individuals with antitrypsin deficiency. The advantage is that this approach will detect many mutants that cause antitrypsin deficiency whilst the existing test detects only the Z allele.

(v) The cell penetrating monoclonal antibodies exploit technology developed at UCL (http://www.thiologics.com) and will be developed to image polymers within the liver of individuals with antitrypsin deficiency. It is possible that they may also be used to deliver polymer blocking antibodies to the livers of individuals with antitrypsin deficiency. If effective then they will be commercialised through UCL Business (UCLB). It is possible that this approach could be developed to target other intracellular disease processes such as the tauopathies.

(vi) Our work has an immediate impact on patient care as we characterise the scientific and clinical significance of antitrypsin deficiency mutations identified as part of clinical service that is now at the Royal Free Hospital (eg. Hepatology 2010; 52: 1078-1088; Am. J. Resp. Cell Mol Biol. 2015 In press). We have also described the prevalence and risk factors for liver involvement in individuals with PiZZ antitrypsin related lung disease (Am. J. Resp. Crit. Care Med. 2013; 187: 502-508).

(vii) If successful circulating antitrypsin polymers would be the first biomarker that is specific for antitrypsin deficiency related liver disease.

(viiI) If one of our therapeutic approaches is successful then this would provide a rational to screen newborn infants for antitrypsin deficiency to prevent them developing established liver disease. This is not currently undertaken in the UK as it is argued that there is no therapeutic benefit from early diagnosis.

Publications

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Briggs AH (2017) Development of the Galaxy Chronic Obstructive Pulmonary Disease (COPD) Model Using Data from ECLIPSE: Internal Validation of a Linked-Equations Cohort Model. in Medical decision making : an international journal of the Society for Medical Decision Making

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Busch R (2017) Genetic Association and Risk Scores in a Chronic Obstructive Pulmonary Disease Meta-analysis of 16,707 Subjects. in American journal of respiratory cell and molecular biology

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Dickens JA (2016) The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-a1-antitrypsin. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

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Elliston ELK (2018) In Vitro Approaches for the Assessment of Serpin Polymerization. in Methods in molecular biology (Clifton, N.J.)

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Exuzides A (2017) Statistical Modeling of Disease Progression for Chronic Obstructive Pulmonary Disease Using Data from the ECLIPSE Study. in Medical decision making : an international journal of the Society for Medical Decision Making

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Faull SV (2017) Electrophoresis- and FRET-Based Measures of Serpin Polymerization. in Methods in molecular biology (Clifton, N.J.)

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Fra A (2016) Polymers of Z a1-antitrypsin are secreted in cell models of disease. in The European respiratory journal

 
Description Chair, Scientific Priorities Committee, 100k Genomes, Department of Health, UK
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact I am Deputy Chair, Scientific Advisory Committee, 100,000 genomes project (2016-). This national programme aims to embed genomics within the NHS
 
Description Alpha-1 Foundation (US) project grant
Amount $199,993 (USD)
Organisation Alpha-1 Foundation 
Sector Charity/Non Profit
Country United States
Start 09/2018 
End 08/2020
 
Description Developing ex vivo structural biology using natural abundance NMR: the role of conformational dynamics in regulating protein metastability
Amount £637,290 (GBP)
Funding ID BB/T002603/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2019 
End 08/2022
 
Title Developing C. elegant that express M and Z antitrypsin and worms that are hypersensitive to siRNA for a screen to detect the proteostasis network following expression of Z antitrypsin 
Description Developing C. elegant that express M and Z antitrypsin and worms that are hypersensitive to siRNA for a screen to detect the proteostasis network following expression of Z antitrypsin 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact We have only just developed this animal model of disease 
 
Title Software developments to our invertebrate automated phenotyping platform 
Description We have made important software developments to our invertebrate automated phenotyping platform (INVAPP). We have supplemented our existing motility screens with the addition of a developmental screen that monitors phenotypes over an extended period - both lend themselves to high-throughput drug screening. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact We have also developed a system permitting a deep phenotyping screen, which monitors activity of individual worms over long periods. We have shown that INVAPP can also work for other protein misfolding disorders such as a C. elegans model of Alzheimer's disease, so the technology has broader applications. We have also used INVAPP to screen for new therapeutics targeting parasitic disease and by this means we have discovered a new anthelmintic chemotype for the treatment of whipworm which affects half a billion people. This work involved collaboration with Prof Kathryn Else (Manchester) and Prof Angela Russell (Oxford). This work has led to one paper (Pubmed ID: 28182663). Data produced in this study has been deposited in the public database PubChem (accession numbers: [SID] 318018055, 318018056, 318018057, 318018058, 318018059 and [AID] 1224898). 
 
Description GSK small molecules to blcok polymeristaion of antitrypsin 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Provided monoclonal antibody, ELISA assay and expertise on the biology of antitrypsin deficiency
Collaborator Contribution Providing small moelcules as tools to block polymerisation in cell modelsof disease
Impact On oging
Start Year 2008
 
Description Licensing of small molecules for antitrypsin deficiency to Biomarin 
Organisation BioMarin Pharmaceutical
Country United States 
Sector Private 
PI Contribution Alpha-1-antitrypsin deficiency is a rare inherited disorder that results in lung and liver disease. While produced in the liver one of its key functions is to protect the lung and other connective tissue from excessive and uncontrolled degradation. In many patients the deficiency of alpha-1-antitrypsin is caused by a genetic missense mutation (e.g. Z alpha-1-antitrypsin) which leads to the production and accumulation of polymers in liver cells. The build-up of polymers leads to liver damage, which in severe cases, requires liver transplantation. There is currently no treatment for alpha-1-antitrypsin deficiency. Research leading to the development of these compounds has been the main scientific interest of Professor David Lomas, Vice-Provost (Health), UCL. Understanding the molecular and cellular mechanisms of alpha-1-antitrypsin deficiency has been the focus of Prof Lomas' work, since his PhD in the early 1990's. More recently, Prof Lomas and his team worked with GlaxoSmithKline as part of the DPAc collaboration scheme to develop a small molecule treatment which will prevent the polymerization of the mutant Z alpha-1-antitrypsin in the liver. Through the very successful collaboration with GSK over five years, the team discovered and developed a number of small molecules which had the desired effects in pre-clinical studies and a number of these were on the path to the clinic. However, in March 2018, the collaboration was terminated by GlaxoSmithKline due to a change in corporate strategy, and all the project IP was assigned to UCL. UCLB have been working alongside Prof Lomas in the identification of a suitable commercial partner to further progress the development of the small molecules to market and ultimately patient benefit.
Collaborator Contribution UCLB has entered into an exclusive licensing agreement with a leading US biotech company. From this partnership, both parties seek to further develop this potential treatment for conditions mediated by alpha-1-antitrypsin polymerization.
Impact None as yet
Start Year 2019
 
Description Small molecules to treat antitrypsin deficiency 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution GSK will undertake a 'black box' screen to look for small molecules to block the polymerisation of mutant antitrypsin. We will help to characterise the hits
Collaborator Contribution GSK will undertake a high throughput screen to look for small molecules to block the polymerisation of mutant antitrypsin. We will help to characterise the hits
Impact GSK will undertake a 'black box' screen to look for small molecules to block the polymerisation of mutant antitrypsin. We will help to characterise the hits
Start Year 2009
 
Description Small molecules to treat antitrypsin deficiency 
Organisation Novartis
Country Global 
Sector Private 
PI Contribution Novartis will undertake a 'black box' screen to look for small molecules to block the polymerisation of mutant antitrypsin
Collaborator Contribution Novartis will undertake a 'black box' screen to look for small molecules to block the polymerisation of mutant antitrypsin
Impact Novartis will undertake a 'black box' screen to look for small molecules to block the polymerisation of mutant antitrypsin
Start Year 2008
 
Title Small molecules to treat alpha-1 antitrypsin deficiency 
Description Small molecules are being developed to stabilise abnormal conformations in alpha-1 antitrypsin in an attempt to develop small molecules to treat this disease. 
Type Therapeutic Intervention - Drug
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Under active development/distribution
Impact This is an ongoing development.