The research goal of this project is to investigate the role of proteinase inhibitor-based drugs in the treatment of cryptosporidiosis. We will take both a biochemical and a molecular biology approach in the investigation. Proteases of sporozoites, one of the invasive forms of the parasite, will be identified and characterized including assignment of mechanistic class, identification of substrate preferences and delineation of inhibitor profile. We will then focus on cysteine and/or serine proteases for three reasons; 1) These enzymes are often active at the time of invasion and may participate in adhesion, rhoptry secretion, moving junction and parasitophorous vacuole formation and remodeling of the host cell cytoskeleton. Some of these may be accessible in the lumen of the gut for inhibitor based systemic chemotherapy. 2) Techniques for cloning and expression of cysteine and serine proteinases have advanced rapidly. These molecular biology techniques will allow us to circumvent the need for large numbers of parasite for enzyme purification, N- terminal protein sequencing and production of antibody to native protein. 3) Recent successful treatment of protozoan infections in animal models with inhibitors of cysteine proteinases indicated that similar treatment in humans will be practical. Following PCR-based cloning of selected proteinases, a proteinase(s) will be expressed to allow detailed kinetic and inhibitor studies using available fluromethyl ketones or their ketone op equivalents. The same inhibitors will be tested to determine their effect on sporozoite invasion and intracellular development in cell culture. Concurrently, other promising compounds will be identified on the basis of their fit with a computer-generated structure of the target proteinase.