Development of small molecule inhibitors to block the polymerisation of Z Alpha-1-Antitrypsin

Alpha-1-Antitrypsin is the most abundant circulating protease inhibitor. The most important deficiency variant is the Z allele (Glu342Lys) which causes the protein to be retained as PAS positive inclusions within the endoplasmic reticulum of hepatocytes. These inclusions predispose the Z Alpha-1-Antitrypsin homozygote to hepatitis, cirrhosis and hepatocellular carcinoma. The resulting lack of circulating Alpha-1-Antitrypsin allows uncontrolled proteolytic digestion and early onset panlobular emphysema. Z Alpha-1-Antitrypsin is retained within hepatocytes as loop-sheet polymers. Point mutations that reduce the polymerisation of Z Alpha-1-Antitrypsin increase secretion of the mutant protein in cell models of disease. We have identified a hydrophobic cavity bounded by the D and E helices in Alpha-1-Antitrypsin that is patent in the native protein but which is obliterated upon polymerisation. The introduction of point mutations with bulky side chains into this cavity (in order to mimic the binding of a small molecule) reduces polymerisation and increases the secretion of Z Alpha-1-Antitrypsin in cell culture without affecting the inhibitory activity against proteolytic enzymes. Thus we believe that this cavity is an ideal target for rational drug design to develop small molecules that can block polymerisation in vitro and in vivo. In collaboration with Ruben Abagyan (Scripps Institute), we have used the novel ICM docking programme to screen 40,000 compounds in silico for lead compounds that may bind to the D/E helix cavity and block polymerisation. We have identified 24 molecules (all less than 500Da) that may bind to this cavity. All the compounds have been tested in vitro and two reduced the polymerisation of Z Alpha-1-Antitrypsin with one compound being almost as effective as the 12mer reactive loop peptide. I now propose to characterise the interaction between this lead compound and Z Alpha-1-Antitrypsin. Further analysis of this interaction will then allow refinement of the lead compound to develop a novel drug to block the polymerisation of Z Alpha-1-Antitrypsin in vitro and in vivo. Such an approach may form the basis of a ‘cure' for the protein overload and liver disease that are associated with the Z allele of Alpha-1-Antitrypsin.

Curriculum Vitae of Andreas Hector

Contact
Dr. Meera Mallya
Cambridge Institute for Medical Research, Wellcome Trust/ MRC Building, Hills Road,
CB2 2XY Cambridge
UK
Telephone: +44 1223 336825
Fax: +44 1223 336827
E-mail: mm342@cam.ac.uk