In Vitro and In Vivo Assessment of FK506 Analogs as Novel Antifungal Drug Candidates

The immunosuppressive effect of the FK506 analogs was reduced compared with that of FK506. (A) The immunosuppressive effect of FK506 on primary CD4⁺ T helper cells was assessed at various concentrations of FK506 with three replicates using flow cytometry. (First row) Lymphocytes were selected within the boundaries from the forward scatter area (FSC-A) and side scatter area (SSC-A); (2nd row) single cells (within the red box) were selected from the forward scatter height (FSC-H) and forward scatter area (FSC-A) for further analysis; (3rd row) primary cultured T cells from mice were stained with LIVE/DEAD cell viability kits, and the percentage of live cells was determined in the presence of various concentrations of FK506; (4th row) CD4⁺ helper T cells were isolated with CD4 T-cell enrichment kits. The assay included a negative-control group with inactivated T cells (NC), a positive-control group with activated T cells (PC), and a vehicle group with activated T cells and a concentration of 0 μg/ml. (B) The cytotoxicity of FK506 and its analogs is shown by the percentage of live cells treated with 0.00001 to 1 μg/ml FK506 and its analogs normalized to the number of live cells treated with vehicle. FK506 at 0.001 μg/ml significantly reduced cell viability by ∼50%, whereas none of the FK506 analogs at the same concentration showed any cytotoxicity. (C) CellTrace Violet (CTV) was used to dye the cells immediately following culture, and intensity levels were measured to detect proliferation after 72 h of drug exposure. The experiment was performed with three replicates for each concentration of each analog compound tested across a range of concentrations. Multiple peaks indicating the proliferation of T cells diminished at concentrations associated with immune suppression. The percentage of proliferating cells within each group was normalized to the number of cells treated with the vehicle. (D) Data from three independent biological groups were used to calculate the IC₅₀ for each compound using nonlinear regression fit analysis. Curves for all compounds displayed higher IC₅₀s compared with the IC₅₀ of FK506, which indicated a significant reduction in immunosuppressive activity. All data are presented as the mean ± standard error of the mean (SEM).

All FK506 analogs, except for 9-deoxo-prolyl-FK506, exhibited dose-dependent antifungal activity against C. neoformans. (A) Disk diffusion halo assays revealed enhanced inhibition of C. neoformans with treatment with FK506 and its analogs. A total of 2 × 10⁷ wild-type (H99 strain) cells were resuspended in top agar and poured onto yeast extract-peptone-dextrose (YPD) solid medium. The disks contained 0.1, 1, or 10 μg of FK506 and its analogs. All the FK506 analogs, except for 9DP-FK506, significantly enhanced halo clearing in a concentration-dependent manner at 37°C. (B) MIC test using 2-fold serially diluted FK506 and FK506 analogs from 15 μg/ml. 9D-FK506, 9D31OD-FK506, and 31OD-FK506 suppressed the growth of nine C. neoformans isolates. (C) MCC1 (frr1::ADE2 mutant) was resistant to 30 μg/ml of FK506, 60 μg/ml of 9D-FK506, 100 μg/ml of 9DP-FK506, 60 μg/ml of 9D31OD-FK506, and 40 μg/ml of 31OD-FK506, whereas its background strain (ade2Δ mutant M049) and H99 cells were susceptible at 37°C. (D) Each C. neoformans strain (the H99 wild-type [WT], cna1Δ, cnb1Δ, and ire1Δ strains) was grown overnight, 10-fold serially diluted, and spotted onto YPD medium with or without 1 μg/ml of the FK506 analogs and 0.2 μg/ml of tunicamycin (TM). (E) Synergism between fludioxonil and the FK506 analogs. A disk diffusion assay was performed with FK506, its analogs (4 μg of FK506 and 31OD-FK506 and 6 μg of 9D-FK506, 9DP-FK506, and 9D31OD-FK506), and fludioxonil at 30°C. DMSO, dimethyl sulfoxide; FDX₁₀ and FDX₁₀₀, 10 and 100 μg fludioxonil, respectively.

All FK506 analogs, except for 9-deoxo-prolyl-FK506, exhibited antifungal activity against C. albicans SC5314. (A) Growth of C. albicans (10-fold serially diluted from an OD of 1.0) on 50% FBS agar containing 3 μg/ml of FK506 or its analogs (5 μg/ml 9D-FK506, 10 μg/ml 9D31OD-FK506, 10 μg/ml 9DP-FK506, or 3 μg/ml 31OD-FK506) at 37°C for 24 h. Growth was suppressed compared with that of the nontreated control. All FK506 analogs, except for 9-deoxo-prolyl-FK506, resulted in reduced growth rates over 1 day. (B) MIC test using 2-fold serially diluted FK506 and FK506 analogs from 10 μg/ml.

All FK506 analogs, except for 9-deoxo-prolyl-FK506, exhibited antifungal activity against A. fumigatus Af293. (A) Growth of A. fumigatus (5,000 spores/2 μl) on YPD containing 1 μg/ml of FK506 or its analogs at 37°C for 3 days. The diameter of each colony differed from that of the nontreated control. All FK506 analogs reduced the growth rate across 3 days, except for 9DP-FK506. (B) MIC test using 2-fold serially diluted FK506 and its analogs from 50 μg/ml.

Calcineurin inhibitors exhibited synergistic antifungal activity with azoles against C. neoformans and C. albicans. (A) Disk diffusion halo assays demonstrated enhanced inhibition of C. neoformans when fluconazole and voriconazole were combined with the FK506 analogs. Wild-type (H99) cells were grown in YPD medium overnight. For each strain, 2.5 × 10⁷ cells/ml were resuspended in top agar (10 ml) and poured onto YPD solid medium (20 ml). Cells were incubated for 48 h at 30 and 37°C. FK506 (2 μg) exhibited synergistic antifungal effects at 37°C when combined with 1 μg of voriconazole or 25 μg of fluconazole. Castor oil solution (5 μl) was used as a negative control. (B) C. albicans (SC5314) cells were grown in YPD medium overnight. The 2-fold serially diluted cells were spotted onto YPD containing 1 μg/ml of FK506, 9-deoxo-FK506 (9D-FK506), 9-deoxo-31-O-demethyl-FK506 (9D31OD-FK506), and 31-O-demethyl-FK506 (31OD-FK506) with or without fluconazole, voriconazole, or itraconazole. (C) FIC assay of FK506, 9D31OD-FK506, and 31OD-FK506 with fluconazole. C. neoformans cells were grown in YPD medium overnight and diluted in liquid RPMI 1640 medium to 0.01 OD unit/ml. The drugs were 2-fold serially diluted from the indicated concentrations. FCZ, fluconazole; VCZ, voriconazole; ITZ, itraconazole.

In vivo efficacy analysis of the FK506 analogs. The C. neoformans cells cultured overnight were washed twice with PBS, and the number of cells was synchronized. (A) In vivo efficacy test using a Galleria mellonella insect model. Amphotericin B (AMB), FK506, and the FK506 analogs were used at 5 mg/kg, and insects were treated three times at 24, 48, and 72 h after inoculation. (B) In vivo efficacy test in a murine model. Cells (5 × 10⁵) were used to infect each mouse via intravenous inoculation. The concentrations of fluconazole (FCZ), FK506, and 9-deoxo-31-O-demethyl-FK506 (9D31OD-FK506) were 3 mg/kg, and the combination group was treated with a mixture of 3 mg/kg of 9D31OD-FK506 with 3 mg/kg of FCZ. The log-rank (Mantel-Cox) test was used for statistical analysis.