Azoles are the most widely used group of antifungals; however, their efficacy is compromised by their fungistatic activity and the selection of resistant strains during treatment. Previous studies have demonstrated roles for MAP kinase and calcium signaling pathways in modulating azole activity. Here we examined the potential role for the evolutionarily conserved pathway mediated by cyclic AMP (cAMP), which is synthesized by the regulated action of adenylate cyclase (encoded by CYR1 in S. cerevisiae and CDC35 in C. albicans) and its associated CAP protein (encoded by SRV2 and CAP1). Relative to wild-type strains, S. cerevisiae and C. albicans strains mutated in these genes were 4 to 67-fold hypersensitive to the azoles fluconazole, itraconazole, or miconazole. These mutants were also 2 to 64-fold hypersensitive to terbinafine and fenpropimorph which, like the azoles, inhibit sterol biosynthesis. Addition of cAMP to the medium reversed the azole hypersensitivity of the cyr1 and cdc35 mutants. An inhibitor of mammalian adenylate cyclase, MDL-12330A, was tested in combination with azoles; a synergistic affect was observed against both azole-sensitive and resistant strains of C. albicans and 5 of 6 non-albicans Candida species. Analysis of cAMP levels in C. albicans after glucose induction in the presence and absence of MDL-12330A confirmed that it acts by inhibiting cAMP synthesis is yeast. RNA hybridization analysis implicated a defect in azole-dependent upregulation of multidrug transporter gene CDR1 in the hypersensitivity of the cdc35 and cap1 mutants. These results provide evidence for a novel mechanism modulating antifungal sensitivity mediated by cAMP signaling; compounds similar to MDL-12330A may be useful in combination with azoles to increase their efficacy as well as to reduce azole resistance.
Full conference title:
The 15 th Congress of the International Society for Human and Animal Mycology
- ISHAM 15th (2003)