The immunosuppresants cyclosporin A, FK506, and rapamycin block signalling events required for T-cell activation. These compounds are natural products and also exhibit potent antimicrobial activity. Previous studies reveal that the mechanisms of drug action are conserved from microorganisms to vertebrates. Within cells, the drugs associate with specific immunophilins: CsA with cyclophilin A and FK506/rapamycin with FKBP12. These complexes form toxic interactions with calcineurin (CsA and FK506) or a kinase homolog that transduces growth promoting signals. Although long touted as potential antifungal agents, little is known about the actions or targets of these immunosuppressants in pathogenic fungi. The investigators propose to examine this in Cryptococcus neoformans, with the ultimate goal of developing selectively toxic agents.
Their Specific Aims are to: (1) Clone the genes encoding homologs of FKBP12 and calcineurin B; cloning of cyclophilin A, calcineurin A, and TOR homologs has been accomplished. (2) Examine the mechanisms of action of the immunosuppressants by gene disruption followed by retesting for sensitivity, site-directed mutagenesis os residues known to confer resistance in other organisms, and characterization of spontaneous resistant mutants. (3) Examine the requirement of these protein targets in C. neoformans virulence, and hence validate them as drug targets, by gene disruption. (4) Identify selectively toxic analogs by screening, and/or generate models of drug interaction with C. neoformans target proteins to aid in the future design of selectively toxic analogs.
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