Microtubules play a crucial role in eukaryotic cell division through formation of the mitotic spindle. They are also major components, in many cell types, of the cytoskeleton and flagella. Consistent with their importance, microtubules are targets for a wide variety of drugs and toxins. The benzimidazoles are notable, however, for their low toxicity to mammalian cells but high activity against helminth and fungal pathogens. Recently, we have demonstrated clinically useful activity of selected benzimidazoles against a protozoan, Giardia lamblia. Pneumocystis carinii pneumonia is a major infectious complication of AIDS for which new chemotherapeutic approaches are needed. P. carinii appears to be a fungus with the drug susceptibility of a protozoan; thus, the anti-P. carinii activity of benzimidazoles warrants examination. Initial studies in a culture model revealed that P. carinii growth is sensitive to selected benzimidazoles at less than 1 microgram/ml. However, to realize the full potential of this drug group, it is important to understand the molecular basis for their selective toxicity. To accomplish this, we will pursue the following specific aims: (1) Clone and characterize P. carinii tubulin genes. Tubulins are the major components of microtubules, and comparisons of tubulin sequences from sensitive and resistant organisms may identify sites important to benzimidazole activity. (2) Characterize tubulin- benzimidazole interactions by mutational analysis. Saccharomyces cerevisiae will be used to genetically define the tubulin-benzimidazole binding site. Partial gene replacement will then be used to permit mutational analysis of P. carinii tubulin in the yeast host. (3) Formulate models for the benzimidazole binding site and evaluate by site-directed mutagenesis. Specific alterations in S. cerevisiae-P. carinii tubulin constructs will be tested for effects on benzimidazole sensitivity. (4) Evaluate anti-P. carinii activity of benzimidazoles in vitro and in vivo. Models for the benzimidazole-tubulin binding site should permit us to rationally select or design derivatives with enhanced selective toxicity. These will be tested in culture and in the rat model. Effects on attachment, morphology, and differentiation will be examined, and attempts will be made to isolate resistant mutants.
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