Antimicrobial Agents and Chemotherapy, March 2000, p. 739-746, Vol. 44, No. 3
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Departments of Medicine,1 Genetics,4 Pharmacology and Cancer Biology,6 and Microbiology,5 Howard Hughes Medical Institute,7 Duke University Medical Center, Durham, North Carolina 27710; College of Medicine, Medical University of South Carolina, Charleston, South Carolina 294252; and Institute of Infectious Diseases and Public Health, University of Ancona, Ospedale Umberto I, 60121 Ancona, Italy3
Received 7 September 1999/Returned for modification 16 November 1999/Accepted 20 December 1999
Cryptococcus neoformans is an opportunistic fungal
pathogen that causes life-threatening infections of the central nervous system. Existing therapies include amphotericin B, fluconazole, and
flucytosine, which are limited by toxic side effects and the emergence
of drug resistance. We recently demonstrated that the protein
phosphatase calcineurin is required for growth at 37°C and virulence
of C. neoformans. Because calcineurin is the target of
potent inhibitors in widespread clinical use, cyclosporine and FK506
(tacrolimus), it is an attractive drug target for novel antifungal
agents. Here we have explored the synergistic potential of combining
the calcineurin inhibitor FK506 or its nonimmunosuppressive analog,
L-685,818, with other antifungal agents and examined the molecular
basis of FK506 action by using genetically engineered fungal strains
that lack the FK506 target proteins FKBP12 and calcineurin. We
demonstrate that FK506 exhibits marked synergistic activity with the
H+ATPase inhibitor bafilomycin A1 via a novel
action distinct from calcineurin loss of function. FK506 also exhibits
synergistic activity with the pneumocandin MK-0991/caspofungin acetate
(formerly L-743,873), which targets the essential
-1,3 glucan
synthase, and in this case, FK506 action is mediated via
FKBP12-dependent inhibition of calcineurin. Finally, we demonstrate
that FK506 and fluconazole have synergistic activity that is
independent of both FKBP12 and calcineurin and may involve the known
ability of FK506 to inhibit multidrug resistance pumps, which are known to export azoles from fungal cells. In summary, our studies illustrate the potential for synergistic activity of a variety of different drug
combinations and the power of molecular genetics to define the
mechanisms of drug action, as well as identify a novel action of FK506
that could have profound implications for therapeutic or toxic effects
in other organisms, including humans.
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