In this thesis, different approaches based on inhibition and interactions studies, have been used to characterize inhibitors of the non-structural protein 3 (NS3) from the hepatitis C virus (HCV). This involves identification of enzyme inhibitory effects and characterization of interaction mechanisms and kinetics, as well as effects on replication in a cell based system and serum protein binding. All this information contributes to HCV drug discovery.
By using an inhibition assay it was possible to evaluate the effects of NS3 protease inhibitors, tested or used in the clinic, on NS3 variants, representing different model systems often used for drug discovery. This study illustrates the importance of accounting for differences in catalytic properties in comparative analyses, for making relevant interpretations of inhibition data. An SPR biosensor-based assay expanded the first study, and provided kinetic and mechanistic information, by direct interaction analyses of the inhibitors. It revealed significant differences between the different genotypes and model systems, and provided data that can be used to better understand the efficacy of inhibitors.
Additionally, novel NS3 protease inhibitors were evaluated with respect to their potential to interfere with protease activity, their sensitivity to resistant mutants and effect on HCV replication. The most potent compounds were also characterized by their bioavailability, solubility and metabolic stability. This provides information for design of improved NS3 protease inhibitors, suggesting potential peptidomimetic structures for the backbone as well as for peptide substituents. These modification strategies allowed inhibitors to be truncated and less peptide-like, still with retained inhibitory effect.
A new strategy for analysis of serum protein binding, of importance for drug distribution was also developed. By defining and using the concept of binding efficiency, serum protein interactions of moderate affinity, as described by rapid kinetics, were characterized. This strategy is also applicable for analysis of low affinity interactions.
Taken together, all these studies provide knowledge and strategies for HCV drug discovery, and by using this information we might take a step closer to the final goal, which is to eradicate HCV.
Uppsala: Acta Universitatis Upsaliensis, 2013. , 67 p.
Danielson, U. Helena, Professor