Understanding HIV-disease is an issue that is at the forefront of medicine and science today. The most effective target in the fight against this disease has been HIV protease (HIV-PR), an essential enzyme in the life cycle of the virus. The use of protease inhibitors have allowed strides to have been made in the more effective treatment of persons infected with HIV, since these drugs bind tightly to the protein and prevent further viral processing. The success of these treatments, however, is halted by the protease's ability to functionally tolerate mutations which confer drug resistance. Clearly, a better understanding of the HIV-PR molecule is needed in order to develop successful treatment strategies in the management of HIV infected patients. The basic goals of my research are to provide an understanding of the functional requirements of the HIV-PR molecule to maintain it's viability, stability, and dynamics. My approach will be to utilize invariant sites in HIV-PR to determine the role that they are playing in this regard. Elucidating the functional role of these residues will provide the basis for the development of strategies which can be used to more effectively halt the activity of the protease while at the same time reducing or eliminating the potential for drug-resistant mutations to occur. The basic strategy to be utilized is that of constructing HIV-PR mutants that vary at the sidechains of interest so as to disrupt their role in the enzyme, and analyze the biophysical properties of the HIV-PR variants as compared to wild type HIV-PR. Techniques to be utilized to undertake these analyses include X-Ray crystallography (in the 2 Angstrom range), differential scanning calorimetry, isothermal titration calorimetry, spectrophotometric enzymatic assays, and NMR analysis. This information will aid in the design of second generation inhibitors.
