Cryptococcus neoformans causes substantial morbidity and mortality for AIDS patients. Because current antifungals are only marginally adequate, our long term objective is to facilitate development of more effective and less toxic antifungals. To accumulate data for rational drug design, the specific aims include probing drug-target interactions for cytochrome P450 lanosterol 14alpha-demethylase (14DM), the drug target for azole antifungals. In vitro mutagenesis and genetic selection will permit and isolation and analysis of 14DM mutants with altered sensitivity to azole antifungals. To help identify and evaluate new drugs and drug targets, the specific aims include developing an in vitro drug screen and evaluating the potential of the C. neoformans NADPH-cytochrome P450 reductase (CPR) as a new drug target.
The specific aims are: (1) Clone and sequence the genes and cDNAs for 14DM and CPR from C. neoformans. Clone and sequence the human 14DM cDNA. (2) Construct Saccharomyces cerevisiae strains in which the C. neoformans or human coding sequences for 14DM and CPR replace the S. cerevisiae coding sequences. In vitro drug screens based on these strains can be used to identify, compare, and contrast agents which inhibit the lanosterol demethylation reaction encoded by genes of C. neoformans, S. cerevisiae, and humans. In addition, these altered strains will serve as reference standards for mutant 14DMs constructed under Aim 3 and may be used in Aim 4 for evaluating the potential of the C. neoformans CPR as a drug target. (3) Screen 14DM mutants, generated in vitro, to identify regions and residues of 14DM involved in azole resistance and demethylase function. Screen azole-resistant clinical isolates of C. neoformans to identify and characterize those with 14DM mutations. (4) Determine if ablation of the C. neoformans CPR gene potentiates the effect of azole antifungals as a step in evaluating the potential of the C. neoformans CPR as a drug target. (5) Examine virulence for selected mutants from Aims 2-4 in a mouse model.
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