Therapeutic antibodies are the standard of care for certain cancers, auto-immune and infectious diseases, but their efficacy decreases when the number of target molecules is too large. The engineering of proteases that specifically catalyze the cleavage of disease-promoting proteins would enable enzyme therapies for a wider range of diseases. HIV protease is a nearly ideal starting point for this purpose because three lines of evidence suggest that this enzyme is unusually evolvable. First, HIV protease evolves rapidly in nature in response to synthetic protease inhibitors. Second, this observed evolvability is consistent with evolutionary theory. Third, the investigator has already employed random mutagenesis and screening to create a variant that is specific for TNF-alpha (target protein implicated in rheumatoid arthritis). He now proposes studies with the following specific aims: 1. to improve the activity and TNF-alpha specificity of the HIV protease variant through a directed evolution process that mimics antibody affinity maturation. 2. to direct the evolution of HIV protease variants specific for the oncogenic protein HER2. 3. to discover the structural mechanisms of adaptive evolution by purifying the wild-type and variant HIV proteases and characterizing their biophysical and kinetic properties in vitro. These experiments are a first step toward a new approach to enzyme therapy. We will demonstrate the unusual evolvability of HIV protease, and better understand why some enzymes are more amenable to protein engineering than others. The high throughput screens developed for these studies could also be used to identify small molecule protease inhibitors.
