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Characterisation of the Z-Pocket for the Treatment of Alpha-1-Antitrypsin Deficiency Œ

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Characterisation of the Z-Pocket for the Treatment of Alpha-1-Antitrypsin Deficiency ÂŒ Impact Objectives • Develop an understanding of serpin polymerisation, and in particular, the defect caused by the common Z variant of alpha-1-antitrypsin • Identify and develop therapeutic agents to rescue Z alpha-1-antitrypsin folding to prevent manifestation of associated lung and liver diseases • Move the findings of Z Factor Ltd through clinical trials to treat the estimated 200,000 people worldwide who are homozygous for the Z mutation Understanding the Z-mutation in antitrypsin Professor James Huntington is the founder of the drug discovery company Z Factor Ltd, which develops novel therapeutic agents to treat alpha-1-antitrypsin deficiency. Here he discusses the potential real- world benefits of his team’s findings Can you begin by mechanism was in operation, but that it was This means that optimisation of small sharing a little about not the only one. The results were published molecule design is unlikely to be achievable how your research in Nature in 2008. by structure-based methods alone. In native career has developed? Z antitrypsin, the Z-pocket is also quite Why does alpha-1-antitrypsin polymerise in dynamic, making it difficult to study. In 1992 I began those with the Z-mutation? working on serpins as a graduate student What is the role of Z Factor Ltd in the at Vanderbilt University in the US. My The Z-mutation only causes a folding defect, research? interest was less in the biology and more so the small amount of native Z antitrypsin in the remarkable conformational changes that does escape the liver cells, in which it is It is very difficult to develop a drug in an that serpins undergo as part of their expressed, is active as an inhibitor of human academic lab. Z Factor provides the funds mechanism. I studied the egg-white protein neutrophil elastase. The key is therefore and also the competencies to discover and ovalbumin and how it changed properties to understand how the Z-mutation slows screen millions of compounds. It utilises when eggs aged, and how antithrombin was normal folding and causes the accumulation contract research organisations instead of conformationally activated by heparin. In of hyper-stable polymers in hepatocytes. The employees, so we can use experts in many 1997, I began a postdoc in Robin Carrell’s mutation is a charge reversal on the stalk different techniques when required. All group in Cambridge, UK to investigate that supports the reactive centre loop, on stages of drug discovery and development how serpins change their shape as part the C-terminus of the protein. The structural are informed by engaging experts in small- of their mechanisms of action. One of effect of the Z-mutation is to slow the folding molecule drug design, and so oral availability our investigations involved how serpins of the C-terminus of the protein, leading and safety is as much a guiding principle polymerised and what could be done to rapid clearance of the long-lived folding as efficacy from the outset. This greatly to prevent it and to treat the resulting intermediate (no secretion) or polymerisation accelerates development and increases our deficiencies. Because antithrombin deficiency (accumulation in addition to no secretion). chances of obtaining a molecule with the causes thrombosis and is sometimes due to We discovered that Z antitrypsin polymerises desired profile. point mutations that cause polymerisation, by a C-terminal domain swap of about 30 I initially focused on antithrombin polymers residues, and that this off-pathway event What are the real-world benefits of this work? with an outstanding postdoc, Masayuki is caused by the Z-mutation destabilising Yamasaki. A stable dimer had been described an element called the BC-barrel. We At the moment there is no effective treatment in patients, and we decided to produce one hypothesised that small molecules could for the disease manifestations of antitrypsin in vitro for crystallisation. The structure stabilise the BC-barrel to allow rapid folding deficiency, 95 per cent of which is caused surprised us all, showing a 50 amino acid of the C-terminus, thereby rescuing folding by the Z-mutation. Chronic obstructive domain swap. This did not fit with the and allowing secretion into the blood. pulmonary disease and cirrhosis are dogma of the ‘loop-sheet’ mechanism irreversible and debilitating. A treatment to where native serpins could swap a small What problems have you thus far faced in slow disease progression would be of huge part of an element called the reactive terms of learning more about the Z-pocket? benefit to those already suffering symptoms, centre loop. Was this an artefact or did it but one that prevents the onset of disease represent how serpins polymerised from The Z-pocket that we identified in a structure would commute the life sentence of people a folding intermediate? We investigated of the polymer is unlikely to be the same homozygous for the Z-mutation. this and concluded that this domain-swap as the pocket in the folding intermediate. www.impact.pub 61 Revolutionising treatment for alpha-1-antitrypsin deficiency Z Factor Limited is a drug discovery company focused on identifying and developing therapeutic agents to treat alpha-1-antitrypsin deficiency. By exploiting recent insights, it is hoped an important and currently unmet medical need can be addressed Alpha-1-antitrypsin is a member of the polymerisation is required to find a means if this mechanism also explained how serpin family of serine protease inhibitors of correction. Z-antitrypsin polymerised (2). The and is one of the most abundant proteins Huntington Group was successful in in the blood. Its principal role is to inhibit Alpha-1-antitrypsin deficiency can be obtaining a Medical Research Council proteases released by immune cells, such caused by many different mutations, but grant on polymerisation, which focused as neutrophil elastase. The mechanism over 95 per cent of cases involve the so- on determining what was responsible for that serpins use can be thought of as a called Z-mutation, Glu342Lys. Around 4 polymerisation of the common Z variant of mousetrap, where the serine protease per cent of people of European descent antitrypsin. They discovered that a different (mouse) bites the reactive centre loop of carry the mutation, and roughly 1 in 2000 domain swap – involving the C-terminal 30 the serpin (bait) followed by springing are homozygous (ZZ). The antitrypsin residues – was the linkage of Z antitrypsin of the trap to crush the protease. This is levels in ZZs are only about 10 per cent polymers (3) - see image right. However, it achieved by a large serpin conformational of normal, and are insufficient to combat was not until the team subsequently solved change involving insertion of the reactive the elastase activity in lungs, leading to the structure of a Z polymer purified from centre loop into a central β-sheet and the chronic obstructive pulmonary disease living cells, that they discovered the detail translocation of the covalently-attached (COPD). In addition, the accumulation which explains why the Z-mutation slows protease to the other pole of the serpin. of polymers in the liver cells that express folding and causes polymerisation. From This conformational rearrangement alpha-1-antitrypsin can cause liver diseases, this, it was clear there was a potential to renders the serpin hyper-stable and such as cirrhosis and cancer. Currently, rescue folding to treat alpha-1-antitrypsin unfolds the protease, leading to a treatments for antitrypsin deficiency are deficiency. stable covalent complex (1). limited to weekly infusions of protein purified from blood plasma, and in severe THE DISCOVERY OF THE Z-POCKET Serpins regulate a wide range of biological cases lung and/or liver transplantation. The structure revealed a special feature, processes, including coagulation and termed the Z-pocket, which explained why inflammation, so diseases generally ensue TREATING ALPHA-1-ANTITRYPSIN polymerisation occurs, but also suggested from serpin deficiencies. Due to the DEFICIENCY that binding of compounds in the pocket complex mechanism of protease inhibition, To understand how serpins polymerise, might be able to fix the defect, thereby serpins are susceptible to mutations that and more specifically, why the Z-mutation rescuing folding and secretion. In order result in a special type of misfolding, causes misfolding of alpha-1-antitrypsin, to test this hypothesis, the team needed known as polymerisation, that reduces the Professor James Huntington and his to utilise the tools of in silico screening to amount of serpin available for protease group have engaged in years of structural identify hits and conduct in vitro studies inhibition and leads to accumulation of and biochemical studies. In 2008, to test their effectiveness. ‘I brought the hyper-stable polymers in secratory cells. they published the first structure of an project to the attention of David Grainger, intact serpin polymer that revealed an with whom I had worked previously on Understanding of the specific mechanisms unexpected domain-swap of over 50 amino another translational venture, XO1 Ltd. involved in serpin misfolding and acids. They then set out to determine Together we were able to raise a round of 62 www.impact.pub Project Insights FUNDING MRC project grant: Structural basis of serpin polymerisation, G0801899, March 2009 – Feb 2012 MRC project grant: Characterisation of the Z-pocket for the treatment of alpha- 1-antitrypsin deficiency, MR/L017431/1, June 2014 – May 2017 Seed phase funding for Z Factor: funding round led by Index Ventures (now Medicxi), Feb 2015 – Feb 2017 Series A funding round of £7m lead by Medicxi, March 2017. CONTACT Professor James A Huntington Project Coordinator T: 01223 763230 E: [email protected] We have identified several compounds that are effective W: http://huntingtonlab.cimr.cam.ac.uk/ www.zfactor.co.uk at rescuing Z antitrypsin folding in vitro and in vivo, PROJECT COORDINATOR BIO and will now develop them further so we can eventually Professor James Huntington obtained a PhD from Vanderbilt University, US conduct clinical trials in 1997 for work on the biophysical characterisation of members of the serpin family of proteins with Peter Gettins.
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