E. Hystolica EhADH2 Enzyme as Anti-Amoebic Target (2007 - Present)

Investigator:  Avelina Espinosa, Roger Williams University 

Abstract:  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. Metronidazole has toxic side effects, neurological complications, and metronidazole-resistant E. histolytica strains have been developed. 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. Three specific aims will help achieve this goal: 1. Identify candidate chemicals with inhibitory capabilities against EhADH2. Test potential inhibitors in vitro using an Escherichia coli AdhE deficient strain, and later in vivo using Entamoeba histolytica trophozoites. Useful candidates will be effective in inhibiting the enzymatic activity of EhADH2 in vitro, killing trophozoites with little or no effect to mammalian enzymes. 2. Define the structure and catalytic function of EhADH2 with the purpose of designing specific anti-amoebic agents. To achieve this goal, the analysis of EhADH2 (wild-type and mutants) in combination with inhibitors, using kinetic, spectrophotometric, computer modeling, and x-ray crystallography will be performed. 3. Determine the toxicity of EhADH2 in mammalian cells. To evaluate the toxicity of these drugs, various concentrations of drugs will be tested in liver and intestine culture cells. EC50 standard procedures will be used.