Cryptosporidium Enzymes as Chemotherapeutic Targets

Petersen, Carolyn

Abstract

Cryptosporidium parvum is a coccidian parasite which causes sever, unremitting diarrhea leading to life-threatening dehydration, malabsorption and cachexia in persons with AIDS. There is currently no effective therapy for cryptosporidiosis. The purpose of this NCDDG program project is to characterize key Cryptosporidium enzymes that play pivotal roles in the life cycle and metabolism of the parasite and to design and identify inhibitor-based drugs that are potent and selective targets of these target enzymes. Our overall strategy is to biochemically active recombinant enzymes in amounts sufficient for detailed analysis of their kinetic properties and inhibitor sensitivities. This empirical drug discovery effort will be coupled with a molecular modeling and computer assisted drug design effort to model the three dimensional structures of the enzymes from their deduced amino acid sequences and to predict candidate inhibitors using molecular graphic techniques. The NCDDG contains three individual projects supported by a material (oocysts and intracellular parasites) and administrative support core. Project 1 will biochemically investigate the enzymes involved in Cryptosporidium purine salvage and will focus initial molecular cloning efforts on the hypothetically key purine salvage enzyme hypoxanthine- guanine-xanthine-phosphoribsyl transferase HGXPRTase). Project 2 will concentrate on the expression of catalytically active recombinant thymidylate synthetase-dihydrofolate reductase (TS-DHFR) which is indispensable for folate synthesis and provides the only source of thymidylate for DNA synthesis. Project 3 will identify and biochemically characterize sporozoite proteinases that have essential functions in the host-parasite relationship. Cysteine and/or serine proteinases identified in these studies will be molecularly cloned, sequenced and eventually expressed. Prototek, Inc. will synthesize or provide peptide inhibitors suggested by the initial inhibitor profiles and by molecular modeling based on the amino acid sequence.