| dc.description.abstract | Cryptosporidium parvum is a water-borne and food-borne apicomplexan pathogen. It is one of the top four diarrheal-causing pathogens in children under the age of five in developing countries, and an opportunistic pathogen in immunocompromised individuals. The preventative measures are not fully effective with, nitazoxanide (NTZ), the only FDA-approved drug for use in immunocompetent individuals. Unlike other apicomplexans, C. parvum lacks Kreb’s cycle and cytochrome-based respiration, thus relying mainly on glycolysis to produce ATP. In this study, we characterized the primary biochemical features of the C. parvum glucose-6-phosphate isomerase (CpGPI) and determined its Michaelis constant towards fructose-6-phosphate (Kvm = 0.309 mM, Vvmax = 31.72 nmol/μg/min). We also discovered that ebselen, an organoselenium drug, was an inhibitor of CpGPI by high-throughput screening of 1,200 known drugs. Ebselen acted on CpGPI as an allosteric noncompetitive inhibitor (ICv50 = 8.33 μM), while complete inhibition of CpGPI activity was not achieved. Although ebselen is useful in studying the inhibition of CpGPI enzyme activity, further proof is needed to chemically and/or genetically validate CpGPI as a drug target. We also identified four drugs as CpHK inhibitors with micromolar level of anti-cryptospordial activities at concentrations nontoxic to the host cells (i.e., hexachlorphene, thimerosal, alexidine dihydrochloride and ebselen with ECv50 = 0.53, 1.77, 8.1 and 165 μM, respectively). The anti-CpHK activity of the four existing drugs provided us new reagents for studying the enzyme properties of the parasite hexokinase. We have previously observed that 2-deoxy-D-glucose (2DG) could inhibit both the enzyme activity of C. parvum hexokinase (CpHK) and the parasite growth in vitro. However, the action and fate of 2DG in C. parvum was not fully investigated. In the present study, we showed that, although 2DG could be phosphorylated by CpHK to form 2DG-6-phosphate (2DG6P), the anti-cryptosporidial activity of 2DG was mainly attributed to the action of 2DG on CpHK, rather than the action of 2DG or 2DG6P on the downstream enzyme CpGPI, nor 2DG6P on CpHK. These observations further supported the hypothesis that CpHK could serve as a drug target in the parasite. | en |
