NOVEL INHIBITORS OF FUNGAL ASPARTIC PROTEINASES

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DESCRIPTION: Immunocompromised patients are susceptible to infection by organisms that are typically cleared by the normal host immune system. Candida albicans (CA) infection leads to several moderate to life threatening or disseminated clinical diseases. Agents that could prevent this infection would help relieve suffering in patients undergoing transplantation, chemotherapy, and fighting AIDS. Proteases secreted by CA aid in penetration through the extracellular matrix to spread infection. These proteases are believed to be virulence factors, and are members of the aspartic proteinase family. It is known that pepstatin, a general inhibitor of aspartic proteinases, will suppress growth of CA. We have recently determined the three-dimensional structure of a related fungal enzyme, yeast proteinase A (YprA) in complex with its natural protein inhibitor, IA-3. This new complex reveals novel features of the inhibition that we proposed to explore further in this project. IA-3 is completely unfolded in solution, as we have established by NMR analysis. By understanding the interactions between YprA and IA-3, we plan to design new inhibitors targeted to the closely related protease from CA. We will achieve this objective through four specific aims. In Aim 1, we will create mutants of the IA-3 to aid in studies of the helix-coil transition and the physical interaction with enzyme through fluorescence energy transfer and continuous-flow micro-mixing procedures. In Aim 2, mutants will be designed to determine critical points of interaction, which could result in simplification of the inhibitor structure. Based on the interactions of the mutants with YprA, we will select some for studies by NMR in both Aims 1 and 2 to determine the dynamics of the unbound state and changes in structure upon binding. In Aim 3 we will make changes in the structure to permit binding to and inhibition of the Candida albicans protease of similar structure and function. In Specific Aim 4 we will exploit the new information provided by the IA-3/YprA complex to aid in design of small molecule inhibitors that achieve specificity through a new mechanism of binding. Compounds developed through this program will be tested against Candida albicans in culture.