The overall goal of this project is to develop new compounds that will exhibit anti-viral activity against the hepatitis C virus. To achieve this objective, we will target the essential processing enzyme, NS3. Based on the unusual substrate specificity at processing points in the viral polyprotein, it should be possible to design and construct selective and effective compounds. Our plan to realize the overall goal consists of the following specific aims: 1. The catalytic domain of HCV NS3 will be expressed to produce adequate amounts of protein for structural and biochemical characterization. Due to problems observed by others, we will attempt the following variations: (a.) The catalytic domain will be subcloned into an E. coli secretory vector to direct the expressed protein to the periplasmic space; (b.) The catalytic domain will be fused to maltose- binding protein or Glutathione-S-transferase, expressed in E. coli, purified by affinity chromatography followed by Factor Xa cleavage; (c.) Mutant sequences will be constructed to substitute surface residues with hydrophilic residues; We will also add blocks of lysine at the N- and C- terminal ends in a further effort to increase solubility; (d.) Larger segments of the HCV genome, including extensions on the N- and/or C- terminal sides, will be expressed; (e.) The baculovirus expression system and the yeast, pichoris pastoris, expression system will also be employed to obtain higher yields of correctly folded, active material. 2. With functional protein in hand, studies on the structural/catalytic properties of HCV NS3 will proceed. We will: (a.) Provide protein to a collaborator for determination of the three- dimensional structure; (b.) Study the catalytic activity in a fluorometric assay designed based on cleavage specificity; (c.) Explore the range of permitted sequence variation on both sides of the cleavage point. This information will contribute to inhibitor design; (d) Study the properties of variants of HCV NS3 derived from different genotypes and subtypes. 3. With an assay established, we can design, construct, and evaluate inhibitors to discover antiviral lead compounds. These new compounds could be used in the crystallographic studies in 2a above. The systems we will explore include: (a.) Small inhibitors such as isocoumarins, chloromethylketones, and boronic acids; and (b.) Small protein proteinase inhibitors, such as BPTI. Although we will not use mutants of BPTI as therapeutic agents, it is likely that information from such protein- protein interacting systems will yield clues to drive inhibitor design.
