Synergistic antifungal activities of bafilomycin A(1), fluconazole, and the pneumocandin MK-0991/caspofungin acetate (L-743,873) with calcineurin inhibitors FK506 and L-685,818 against Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic fungal pathogen that causes lif e-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 demons trated that the protein phosphatase calcineurin is required for growth at 3 7 degrees C and virulence of C. neoformans, Because calcineurin is the targ et 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 calcineuri n inhibitor FK506 or its nonimmunosuppressive analog, L-685,818, with other antifungal agents and examined the molecular basis of FK506 action by usin g genetically engineered fungal strains that lack the FK506 target proteins FKBP12 and calcineurin, We demonstrate that FK506 exhibits marked synergis tic activity with the H(+)ATPase inhibitor bafilomycin A(1) via a novel act ion distinct from calcineurin loss of function. FK506 also exhibits synergi stic activity with the pneumocandin MK-0991/caspofungin acetate (formerly L -743,873), which targets the essential beta-1,3 glucan synthase, and in thi s case, FK506 action is mediated via FKBP12-dependent inhibition of calcine urin. Finally, we demonstrate that FK506 and fluconazole have synergistic a ctivity 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 illustra te the potential for synergistic activity of a variety of different drug co mbinations 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 pr ofound implications for therapeutic or toxic effects in other organisms, in cluding humans.