Viral proteases are often considered to be attractive drug targets because of their crucial function in the viral replication machinery. In order to increase our knowledge of these important targets and to contribute to the discovery and development of new antiviral drugs, the proteases from hepatitis C virus (HCV) and human cytomegalovirus (HCMV) have been produced and their interactions with inhibitors and fragments have been characterized, using enzyme inhibition and SPR biosensor based interaction assay. The structure activity relationships and the resistance profiles of a series of HCV NS3 protease inhibitors based on either P2 proline or phenylglycine residues were analyzed using wild type genotype 1a and the major resistant variants A156T and D168V. The observed susceptibility to substitutions associated with these resistance variants was concluded to depend on the P2 and the P1 residue, and not only on the P2 residue as previously had been suggested. In order to be able to evaluate how the potency of inhibitors is affected by genetic variation, their effect was evaluated on wild type NS3 from genotype 1a, 1b and 3a as well as on the resistant variant R155K from genotype 1a. To enable a comparison of the inhibitory effect on the enzyme variants, the compounds were analyzed under conditions optimized for each variant. VX-950 was found to be the least susceptible compound to resistance and genetic variation. A more detailed analysis showed that the kinetic and mechanistic features of the inhibitors were significantly different for the different genotypes. The reversible non covalent macrocyclic inhibitor ITMN 191 was revealed to have favorable kinetics for all three genotypes. This is an advantage for the design of broad spectrum drugs. A fragment based procedure for identifying and validating novel scaffolds for inhibitors of HCMV protease was established. It identified fragments that may serve as starting points for the discovery of effective inhibitors against this challenging target.   The procedures developed for the evaluation and identification of novel HCV NS3 and HCMV protease inhibitors have contributed to a deeper understanding of protease-inhibitor interactions that is expected to have an impact on the design of novel antiviral drugs.

Viral proteases are important targets for anti-viral drugs. Discovery of protease inhibitors as anti-viral drugs is aided by an understanding of the interactions between viral protease and inhibitors. This thesis addresses the characterization of protease-inhibitor interactions for application to drug discovery and model system development. The choice of a relevant target is essential to molecular interaction studies. Therefore, full-length NS3 protein of hepatitis C virus (HCV) was obtained, providing a more relevant target and a better model for the development of HCV protease inhibitors. In addition, resistance to anti-viral drugs, a serious problem in the treatment of AIDS, prompted the investigation of resistant variants of human immunodeficiency virus (HIV) protease. Drug resistance was initially explored by characterization of the interactions between a series of closely related inhibitors and resistant variants of HIV protease, using an inhibition assay to determine the inhibition dissociation constants (Ki). The relationship between structure, activity and resistance profiles was not clarified, indicating that the effect of structural changes in the inhibitors and the protease are not predictable and must be analyzed case wise. It was proposed that additional kinetic characterization of the interactions was required and a biosensor-based method allowing for determination of affinity, KD, and interaction rate constants, kon and koff, was adopted. The increased physiological relevance of this method was confirmed, and the affinity data have better correlation with cell culture data. In addition, interactions between clinical inhibitors of HIV protease and enzyme variants indicate that increased dissociation rates (koff) are associated with the development of resistance. Thermodynamic characterization of the interactions between HIV-1 protease and clinically relevant inhibitors revealed distinct energetic characteristics for inhibitors. The resolution of the energetics of association and dissociation identified an inhibitor with unique interaction characteristics and confirmed the validity of using this method for further characterization of molecular interactions. This work resulted in the development of model systems for the analysis of kinetics, resistance and thermodynamic characteristics of protein-inhibitor interactions. The results give increased understanding of the biomolecular interactions and can be applied to drug discovery.