IN VITRO ASSAY FOR (1,3)B-GLUCAN SYNTHASE INHIBITORS

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The fungal cell wall protects the organism against a hostile exterior environment and relays signals for invasion and infection of a likely animal or human host. The wall affords a clear and discernible difference between fungi and their hosts, providing an experimental target for antifungal antibiotics. A key step in fungal wall assembly is the synthesis of (1,3)beta-linked glucan and its subsequent incorporation into the cell wall. The observation that humans lack (1,3)beta-glucan makes (1,3)beta-glucan synthesis an attractive target for antifungal antibiotics. In Phase I, a rapid, high throughput in vitro enzyme assay suitable as a screen to detect inhibitors of (1 ,3)beta-glucan synthase activity of Neurospora crassa was developed. The Specific Aims of this Phase II proposal are: 1. Development of optimum in vitro conditions for the assay of (1,3)beta- glucan synthase of Candida albicans and Aspergillus fumigatus. 2. Development of a semi-automated in vitro assay for (1,3)beta-glucan synthase activity. 3. Screening of bona fide samples for enzyme inhibitors. 4. Follow-up testing of positive samples using whole-cell assays. The development of a high throughput semi-automated in vitro assay of (1,3)beta-glucan synthase activity of human pathogenic fungi will permit the mass screening of samples for enzyme inhibitors. PROPOSED COMMERCIAL APPLICATION: This assay will permit the screening of compounds on a large scale for inhibitors of fungal (1,3)beta-glucan synthase using human pathogens as enzyme sources. Antifungal drugs for human therapeutic use have significant market potential.