Cryptosporidium parvum metalloaminopeptidase inhibitors prevent in vitro excystation

Cryptosporidium parvum arginine aminopeptidase (RAP) was studied during in vitro excystation. Specific RAP inhibitors were identified by using C. parvum extracts. Amastatin, a series of α-aminoboronic acids, and the chelating agents EDTA and 1.10-phenanthrolene, but not endoproteinase inhibitors, blocked enzymatic activity. RAP inhibitors found to be effective in soluble enzymatic assays were then studied for their effect on in vitro excystation. 1,10-Phenanthrolene, amastatin, and 11-boronorleucine (pinacol) inhibited excystation by 84, 57, and 61%, respectively, compared with solvent-treated control oocysts. Sporozoites remained viable within the oocyst an determined by propidium iodide and fluorescein diacetate dye uptake, suggesting that α-aminoboronic acids were not directly lethal to the parasite.