Enzymatic properties of hepatitis C virus NS3 serine protease and bio-engineering of serine protease inhibitors (serpins) against the NS3 protease and elastase

• Main Author: Po, Addy
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/14043

Hepatitis C virus (HCV) has infected millions of people worldwide and emerged as a global health crisis. HCV NS3 serine protease domain (aa:1027-1218) of the HCV polyprotein, is required for the processing and maturation of the HCV viral non-structural (NS) proteins. As an essential protease for viral replication, HCV NS3 protease has been considered as a strategic target for anti-HCV drug development. This thesis investigates the designs and inhibitory properties of novel serine protease inhibitor (serpin) variants against the HCV NS3 protease. First, the substrate specificity of NS3 protease is evaluated for the purpose of generating protein-based inhibitors to target it. Data from kinetic experiments suggested that NS3 protease activity is greatly enhanced with the addition of its NS4A cofactor and that the three key residues in the substrate necessary for efficient recognition and cleavage by NS3 protease are cysteine in P1, serine/alanine in P'1 and aspartic/glutamic acids in P6 position. This information was used to bio-engineer 5 (serpins) variants based on α₁-Antitrypsin (α₁-AT) scaffold. Variants of α₁-AT contained different mutations in the serpin Reactive Site Loop (RSL) involving the P1, P4, P6 & P'1 positions. The biological activities and the newly gained specificities of the ai-AT and its variants were tested mainly by performing SDS-stable complex formation studies and enzyme kinetic experiments directed against NS3 protease and elastase. Results show that NS3 protease was able to form a 72 kDa SDS-stable complex with α₁-AT and 4 of its variants. Taken together, the ability of α₁-AT and its variants to interact and form SDS-stable complexes with NS3 protease may represent an entry point for a novel class of protein-based HCV NS3 protease inhibitors.