Entamoeba histolytica, the agent of amebic dysentery and amebic liver abscess, is the third leading cause of death from parasitic diseases worldwide. Invasive E. histolytica infection is treated with the drug metronidazole, but concerns about the mutagenic effects of metronidazole, and the development of metronidazole resistance in a number of organisms, has fueled a search for alternative agents to treat amebiasis. E histolytica lack mitochondria, and obtain energy from the fermentation of glucose to CO2, acetate and ethanol. This glycolytic pathway is an attractive target for anti-amebic chemotherapy because it is essential for parasite existence, and it appears to utilize several enzymes lacking from most other eukaryotic organisms. Recently, we isolated the native protein, and cloned the gene encoding a key enzyme in this pathway, a bifunctional NAD-dependent alcohol dehydrogenase/acetyl-coA reductase responsible for the conversion of acetyl-coA to acetaldehyde and acetaldehyde to ethanol. This enzyme, which we have designated E. histolytica alcohol dehydrogenase 2 (EhADH2), has a unique structure containing both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) domains, and does not have any homologues among known eukaryotic enzymes. The critical position of the EhADH2 enzyme in the amebic glycolytic pathway, and its fundamental differences from human ADH and ALDH enzymes, appear to make it an ideal target for chemotherapy. The goal of this proposal is to identify specific inhibitors of the EhADH2 enzyme. We will accomplish this by studying the structure and function of the EhADH2 enzyme at the molecular level to learn about this new class of enzymes, and to provide the data base necessary for the rational design of inhibitors of the EhADH2 molecule. At the same time, we will develop an assay system which will allow us to rapidly, simply, and inexpensively screen compounds for EhADH2 inhibitory activity. Inhibitors identified by this screen, and by molecular modeling, will then be assayed for their effects on E. histolytica growth and virulence. Compounds capable of selectively inhibiting the EhADH2 enzyme could represent a new class of agents effective against certain anaerobic protozoa and some anaerobic bacteria.
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