Immunosuppressive and nonimmunosuppressive cyclosporine analogs are toxic to the opportunistic fungal pathogen Cryptococcus neoformans via cyclophilin-dependent inhibition of calcineurin

Abstract

Cyclosporine (CsA) is an immunosuppressive and antimicrobial drug which, in complex with cyclophilin A, inhibits the protein phosphatase calcineurin. We recently found that Cryptococcus neoformans growth is resistant to CsA at 24 degrees C but sensitive at 37 degrees C and that calcineurin is required for growth at 37 degrees C and pathogenicity. Here CsA analogs were screened for toxicity against C. neoformans in vitro. In most cases, antifungal activity was correlated with cyclophilin A binding in vitro and inhibition of the mixed-lymphocyte reaction and interleukin 2 production in cell culture. Two unusual nonimmunosuppressive CsA derivatives, (gamma-OH) MeLeu(4)-Cs (211-810) and D-Sar (alpha-SMe)(3) Val(2)-DH-Cs (209-825), which are also toxic to C. neoformans were identified. These CsA analogs inhibit C. neoformans via fungal cyclophilin A and calcineurin homologs. Our findings identify calcineurin as a novel antifungal drug target and suggest nonimmunosuppressive CsA analogs warrant investigation as antifungal agents.

FIG. 1

CsA is toxic to C. neoformans at 37°C but not at 24°C. C. neoformans H99 was grown in RPMI 1640 medium with the indicated concentrations of CsA for 72 h at 24, 30, and 37°C. Growth was assayed by determining the OD₆₀₀, and three independent experiments were done.

FIG. 2

C. neoformans expresses [³H]CsA binding activities. CsA-binding protein activity was measured by the LH-20 drug binding assay. (A) Tritiated CsA alone (squares) or incubated with purified S. cerevisiae cyclophilin A protein (diamonds) was subjected to Sephadex LH-20 chromatography. Fractions were analyzed by scintillation counting, and the percentages of total counts per minute were plotted. (B) Protein extracts from the C. neoformans wild-type CPA1 CPA2 strain H99 (squares), cpa1 mutant strain PW67 (diamonds), cpa2 mutant strain PW71 (circles), and cpa1 cpa2 mutant strain PW62 (triangles) were subjected to Sephadex LH-20 chromatography. Fractions were analyzed by scintillation counting, and the percentages of total counts per minute were plotted. Elution volume is given in milliliters.

FIG. 3

Structures of CsA and CsA analogs 211-810 and 209-825. The structures of CsA and two nonimmunosuppressive CsA analogs are depicted. The upper half of the CsA ring binds cyclophilin A, and the effector surface of CsA that is exposed to the solvent in the cyclophilin A-CsA complex and which interacts with calcineurin is the lower half of the ring.

FIG. 4

CsA and CsA analogs promote cryptococcal cyclophilin A binding to calcineurin. Binding assays with purified cryptococcal cyclophilin A immobilized on chitin beads were performed with equal amounts of protein extract from serotype A strain H99 (A) and serotype D strain JEC21 (B) in the absence (−) or presence of 100 μM CsA and CsA analogs (γ-OH) MeLeu⁴-Cs (211-810) and D-Sar (α-SMe)³ Val²-DH-Cs (209-825). Bound proteins were eluted and fractionated by SDS-polyacrylamide gel electrophoresis, and the calcineurin A catalytic subunit was detected by an overlay blot with ¹²⁵I-calmodulin. The migration position of the CNA1 protein is indicated by an arrow. Total protein extracts from serotype A and D CNA1 wild-type and cna1 mutant strains served as controls for the identity and electrophoretic mobility of the calcineurin A catalytic subunit CNA1.