There are many O1s in AIDS patients which cause debilitating diarrhea. Cryptosporidium parvum is one of these. The purpose of this application is to explore the polyamine pathway of C. parvum for its potential as a chemotherapeutic target. Our consortium has shown that within the Apicomplexa, C: parvum has a type of polyamine metabolism usually found only in photosynthetic eukaryotes and certain bacteria. Specifically, using biochemical and molecular techniques, this proposal will explore the two key enzymes in this pathway for their ability to serve as Cryptosporidium-specific targets. They are the biosynthetic, forward- directed enzyme arginine decarboxylase (ADC), and athe back-converting, regulatory enzyme spermidine/spermine N1-acetyltransferase (SSAT). Project 1: Molecular Analyses will be directed by Drs. Jan Keithly, PI, and Guan Zhu. They will isolate, clone and characterize the ADC and SSAT genes; confirm their identity by sequencing; study transcription of these genes in the presence and absence of enzyme inhibitors; and test by site- directed mutagenesis the effect of inhibitors upon protein expression. Project 2: Biochemical Analyses will be directed by Dr. Nigel Yarlett, in collaboration with Drs. Patric Woster who will synthesize ADC and SSAT inhibitors, and Dr. Steve Upton who will test these compounds in vitro and in vivo. Dr. Yarlett will characterize each of the enzyme kinetic and drug-binding properties, and will test the effect of inhibitors upon them. In this application we document this unexpected pathway with biochemical, pharmacologic and molecular evidence, and show its potential exploitation for anti-cryptosporidial chemotherapy.
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