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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI032433-03
Application #
2067319
Study Section
AIDS and Related Research Study Section 4 (ARRD)
Project Start
1992-05-01
Project End
1995-04-30
Budget Start
1994-05-01
Budget End
1995-04-30
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Allegheny University of Health Sciences
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19129
Edlind, T D; Henry, K W; Metera, K A et al. (2001) Aspergillus fumigatus CYP51 sequence: potential basis for fluconazole resistance. Med Mycol 39:299-302
Katiyar, S K; Edlind, T D (2001) Identification and expression of multidrug resistance-related ABC transporter genes in Candida krusei. Med Mycol 39:109-16
Henry, K W; Nickels, J T; Edlind, T D (2000) Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors. Antimicrob Agents Chemother 44:2693-700
Henry, K W; Cruz, M C; Katiyar, S K et al. (1999) Antagonism of azole activity against Candida albicans following induction of multidrug resistance genes by selected antimicrobial agents. Antimicrob Agents Chemother 43:1968-74
Katiyar, S K; Edlind, T D (1997) In vitro susceptibilities of the AIDS-associated microsporidian Encephalitozoon intestinalis to albendazole, its sulfoxide metabolite, and 12 additional benzimidazole derivatives. Antimicrob Agents Chemother 41:2729-32
Cruz, M C; Edlind, T (1997) beta-Tubulin genes and the basis for benzimidazole sensitivity of the opportunistic fungus Cryptococcus neoformans. Microbiology 143 ( Pt 6):2003-8
Edlind, T; Katiyar, S; Visvesvara, G et al. (1996) Evolutionary origins of Microsporidia and basis for benzimidazole sensitivity: an update. J Eukaryot Microbiol 43:109S
Edlind, T D; Li, J; Visvesvara, G S et al. (1996) Phylogenetic analysis of beta-tubulin sequences from amitochondrial protozoa. Mol Phylogenet Evol 5:359-67
Li, J; Katiyar, S K; Edlind, T D (1996) Site-directed mutagenesis of Saccharomyces cerevisiae beta-tubulin: interaction between residue 167 and benzimidazole compounds. FEBS Lett 385:7-10
Katiyar, S K; Edlind, T D (1996) Entamoeba histolytica encodes a highly divergent beta-tubulin. J Eukaryot Microbiol 43:31-4

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