Benzimidazoles, widely used as anthelmintic agents, inhibit in vitro growth of the AIDS-associated pathogen Pneumocystis carinii. The derivative albendazole has therapeutic and prophylactic activity in a mouse model of P. carinii infection, although high doses are required. In vitro benzimidazole activity extends to Cryptococcus neoformans and Histoplasma capsulatum, two additional fungi associated with AIDS. Importantly, the activity is cidal, as demonstrated with C. neoformans. Understanding the molecular basis for this activity may lead to the development of clinically more active derivatives or to the discovery of novel microtubule-targeted agents. The presumed benzimidazole target, the microtubule subunit beta-tubulin, has been characterized at the gene level from P. carinii, C. neoformans, H. capsulatum, and additional organisms. An albendazole-resistant C. neoformans mutant was isolated and sequence analysis confirmed that beta-tubulin is the target. Amino acids involved in benzimidazole activity have begun to be identified; the role of one residue was directly tested by site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin. The long term goal of this proposal is to develop new treatments for AIDS- associated fungal infections by targeting microtubules. While benzimidazoles represent a promising lead, it is apparent that a more basic understanding is needed of benzimidazole-microtubule interactions, and of cellular mechanisms regulating polymerization and depolymerization of the microtubule-based mitotic spindle.
Our Specific Aims are to: (I) Define benzimidazole-microtubule interactions by genetic analysis. Mutants of C. neoformans and S. cerevisiae/pc (encoding hybrid S. cerevisiae/P. Carinii beta-tubulin) resistant to specific derivatives will be selected for and characterized. Site-directed mutagenesis of S. cerevisiae and S. cerevisiae/pc will then be employed to define the role of specific amino acids; this information should permit modelling of benzimidazole-tubulin interaction. (2) Examine in vivo activity of benzimidazoles in mouse models of P. carinii and H. capsulatum infection. Parenteral administration of water-solubilized derivatives will be explored, since poor oral absorption and metabolism by the liver represent the primary obstacles to therapeutic activity. Development of resistance in vivo will also be examined. (3) Characterize cellular factors and tubulin peptides regulating microtubule polymerization in S. cerevisiae. Transfection with plasmids carrying alpha- or beta-tubulin genes is lethal, presumably due to disruption of tubulin-microtubule equilibrium. This effect will be exploited to identify a minimal inhibitory tubulin peptide. S. cerevisiae genes will be selected for that overcome growth inhibition due to benzimidazoles or due to incubation of cold- or heat- sensitive beta-tubulin mutants at their restrictive temperatures. This will test the hypothesis that microtubule-targeted drugs mimic the action of cellular factors induced before or after mitosis.
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