Defining the effect of small molecules that block the polymerisation of Z alpha-1 antitrypsin

Alpha-1 antitrypsin is the most abundant circulating proteinase inhibitor. The Z mutation (Glu342Lys) causes the protein to be retained as ordered polymers within the endoplasmic reticulum of hepatocytes. This causes liver disease whilst the lack of circulating protein predisposes the PI*Z homozygote to early onset emphysema. Strategies that block the formation of polymers are likely to be effective in preventing and treating the liver disease associated with Z alpha-1 antitrypsin. An allosteric pocket in the structure of alpha-1 antitrypsin has been identified that is an ideal target for small molecules to block polymerisation. An in silico screen was undertaken to identify small molecules that can bind to this pocket and block the polymerisation of Z alpha-1 antitrypsin in vitro. These small molecules reduce the accumulation of Z alpha-1 antitrypsin by 70% in a Hepa1a cell model of disease. However they do not increase secretion. I now propose to use this fellowship to dissect the mechanism by which these and other small molecules mediate their effect. Therefore, the project has the following specific aims: (i) to determine whether the small molecules identified in the screen can affect the generation, accumulation and clearance of polymers. (ii) to assess whether the small molecules can reduce the endoplasmic reticulum overload response (NF-kB, BiP, GRP94, PDI, eIF2?-P, CHOP, XBP1 and ATF6) that is associated with the retention of Z alpha-1 antitrypsin within hepatocytes and (iii) to use chemical and genetic inhibitors to determine if the small molecules accelerate the clearance of Z alpha-1 antitrypsin monomer or polymers via the proteosomal pathway or by autophagy. These experiments will be undertaken in COS-7 cell models that transiently express wildtype and Z alpha-1 antitrypsin. I will also use cell lines (CHO tet-on) that I have developed that conditionally express wildtype protein and the Z and His334Asp alpha-1 antitrypsin mutants that are associated with polymerisation and liver disease. In addition I will use primary hepatocyte-like cell lines that we have derived from skin biopsies from patients who are PI*Z homozygotes.

Curriculum Vitae of Adriana Ordóñez González

Adriana Ordóñez is a post-doctoral student at the Cambridge Institute for Medical Research at the University of Cambridge's Department of Medicine, UK. In 2009 she completed her European PhD in Medicine at the University of Murcia, Spain. Her research towards a PhD was supported by a prestigious grant from the Spanish Ministry of Health. In 2005, Dr Ordóñez was awarded her undergraduate degree in Biology by the University of Murcia, Spain. In addition to other prizes, Dr Ordóñez won the Award for Best Communication presented at the National Congress of the Spanish Society of Thrombosis and Haemostasis in 2008.

Dr Ordóñez's areas of research include the structure and role of antithrombin and serpins, which has resulted in the co-authorship of numerous publications. Her current eALTA research project "Defining the effect of small molecules that block the polymerisation of Z alpha-1 antitrypsin" focuses on elucidating the mechanisms by which small molecules block the polymerisation of Z alpha- 1 antitrypsin in vitro and describe their down-stream effects.

Contact 
Adriana Ordóñez González
Department of Medicine,
University of Cambridge,
Cambridge Institute for Medical Research,
Wellcome Trust/ MRC Building
CB2 2XY Cambridge, UK