Proteins possess great pharmaceutical potential because their molecular recognition properties are often superior to those of small molecules. Antibodies with high affinity and specificity for almost any target molecule can be produced, but their efficacy as drugs decreases when the number of targets (tumor cells or infectious pathogens) is large. Enzymes that catalyze the inactivation of disease-promoting molecules would be more effective. Our long-term goal is use proteases as drugs, rather than as drug targets. We have chosen HCV protease as a starting template because its natural history suggests unusual evolvability. Collaborators Allison Dennis and Gang Bao have developed a versatile protease-activated molecular switch. We have developed a semi-automated high throughput screen to assess the catalytic activity of individual clones. Here we describe the evolution of hypermorphic HCV protease variants in three rounds of mutagenesis and screening. The most active clone, in combination with the screen and molecular switch, will be used to direct the evolution of variants with altered specificity.

Extended Abstract Status: Not Uploaded