AIDS-related fungal infections are important targets for reduction of mortality and improvement in the quality of life for people living with AIDS. Each new generation of azole drugs, such as fluconazole, has succumbed to emergent cross resistance. Natural products are represented among clinically useful antifungal agents. Marine invertebrates, particularly Porifera (sponges), produce chemically diverse libraries of natural products, some of which possess useful antifungal activity. The general goal of this program is to find and identify small molecules from marine organisms that are active against fluconazole-resistant strains of Candida albicans and inherently fluconazole-resistant non-albicans species, including Candida glabrata and Candida krusei and use these as prototype leads for antifungal drugs. We plan to prepare and screen extracts for antifungal agents using a mechanism-selective approach that may be useful in identifying new leads for antifungal therapies. New emphases in this program are mechanism based screens and the systematic evaluation of compounds with unique mechanisms of action, including inhibition of fungal sphingolipid biosynthesis, that complement current therapies and intervene at strategic points in fungal cell metabolism or life-cycle. Active components will be isolated by a combination of solvent-partitioning, chromatography, liquid-liquid centrifugal counter current chromatography and other techniques. The in vitro antibiotic susceptibilities of pathogenic fungi will be evaluated in a panel of fluconazole-resistant fungi. Selected leads will be advanced to in vivo evaluation in murine models of Candida albicans, Cryptococcus neoformans and Candida glabrata. The structures of novel compounds will be determined by a combination of spectroscopic techniques including mass spectrometry, nuclear magnetic spectroscopy, circular dichroism and X-ray crystallography. Absolute stereochemistry of chiral molecules will be determined using a combination of chiroptical techniques and chemical degradation. Derivatives of existing leads, including the C. glabrata-specific dimeric sphingolipid, oceanapiside, will be synthesised de novo or by semi-synthetic modification to prepare limited libraries of analogs for structure-activity studies. Optimized leads identified from those libraries will be advanced to in vitro and in vivo trials. ? ? ?
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