This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The intestinal protozoan Entamoeba histolytica is a major cause of morbidity and mortality worldwide, causing fifty million cases of diarrhea and one hundred thousand deaths per year. Amoebiasis is primarily treated with metronidazole. Limitations of metronidazole include toxic side effects, neurological complications, and the appearance of metronidazole-resistant E. histolytica strains. These concerns have prompted the search for alternative therapeutic agents. One promising approach is the development of novel anti-metabolites. Entamoeba histolytica lacks mitochondria and obtains its energy from the fermentation of glucose. Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2), a bifunctional enzyme with both aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) activities, constitutes a key enzyme in this pathway. EhADH2 is required for the growth and survival of E. histolytica, suggesting this enzyme could be a target for new anti-amoebic drugs. The goal of this study is to search for alternative non-toxic drugs that inhibit the growth of E. histolytica trophozoites, by specifically affecting the EhADH2 enzyme. Animal models will be used to determine the toxicity of these drugs in humans.
Three specific aims will help achieve this goal: 1. Identify candidate chemicals that inhibit EhADH2. First, test potential inhibitors in vitro using an Escherichia coli AdhE deficient strain, and later in vivo using Entamoeba histolytica trophozoites. Useful candidates should inhibit the enzymatic activity of EhADH2, killing trophozoites with little or no effect to mammalian enzymes. 2. The selected chemicals from aim 1 will be further analyzed in vivo for safety in animal models. To evaluate the toxicity of these drugs, various concentrations of drugs will be tested in animal models (SCID mice). LD50 standard procedures will be used. 3. Further analysis of EhADH2 will aid in designing anti-amoebic agents. EhADH2 (wild-type, mutants) will be analyzed with identified and designed inhibitors, using kinetic, spectrophotometric, computer modeling, and x-ray crystallography.
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