Antifungal Activities of 4″,6″-Disubstituted Amphiphilic Kanamycins

Abstract

Amphiphilic kanamycins derived from the classic antibiotic kanamycin have attracted interest due to their novel bioactivities beyond inhibition of bacteria. In this study, the recently described 4″,6″-diaryl amphiphilic kanamycins reported as inhibitors of connexin were examined for their antifungal activities. Nearly all 4″,6″-diaryl amphiphilic kanamycins tested had antifungal activities comparable to those of 4″,6″-dialkyl amphiphilic kanamycins, reported previously against several fungal strains. The minimal growth inhibitory concentrations (MICs) correlated with the degree of amphiphilicity (cLogD) of the di-substituted amphiphilic kanamycins. Using the fluorogenic dyes, SYTOX^TM Green and propidium iodide, the most active compounds at the corresponding MICs or at 2×MICs caused biphasic dye fluorescence increases over time with intact cells. Further lowering the concentrations to half MICs caused first-order dye fluorescence increases. Interestingly, 4×MIC or 8×MIC levels resulted in fluorescence suppression that did not correlate with the MIC and plasma membrane permeabilization. The results show that 4″,6″-diaryl amphiphilic kanamycins are antifungal and that amphiphilicity parameter cLogD is useful for the design of the most membrane-active versions. A cautionary limitation of fluorescence suppression was revealed when using fluorogenic dyes to measure cell-permeation mechanisms with these antifungals at high concentrations. Finally, 4″,6″-diaryl amphiphilic kanamycins elevate the production of cellular reactive oxygen species as other reported amphiphilic kanamycins.

Keywords: Cryptococcus neoformans; SYTOXTM green; amphiphilic kanamycin; antifungal; kinetic membrane permeabilization; propidium iodide.

Figure 1

Structure of aminoglycosides and selected kanamycins (AKs).

Figure 2

Structures of SYTOX^TM green and propidium iodide (PI).

Figure 3

Structure of selected AKs.

Figure 4

Relationship of the water/1-octanol distribution coefficient (cLogD) vs. minimal growth inhibitory concentration (MIC).

Figure 5

Phase contrast (left panels ) and fluorescent (right panels) images of C. neoformans H99. (A) and (B): Images of cells treated with SYTOX^TM green alone; (C) and (D): Images of cells treated with SYTOX^TM green and 1% Triton X-100; (E) and (F): Images of cells treated with SYTOX^TM green and compound 7 (1×MIC); (G) and (H): Images of cells treated with SYTOX^TM green and compound 11 (1xMIC); (I) and (J): Images of cells treated with SYTOX^TM green and compound 13 (256 μg/mL).

Figure 6

Kinetic membrane permeabilization of C. neoformans H99. (A) Cells treated with 4″,6″-diaryl AKs using SYTOX^TM green; (B) cells treated with 4″,6″-diaryl AKs using PI; (C) cells treated with 4″,6″-dialkyl AKs using SYTOX^TM green; (D) cells treated with 4″,6″-dialkyl AKs using PI. The figure with standard deviation is available in Supplementary Materials.

Figure 7

Kinetic membrane permeabilization of C. neoformans H99 using varied concentrations of AKs. (A) Cells treated with compound 7 using SYTOX^TM green; (B) cells treated with compound 7 using PI; (C cells treated with compound 10 using SYTOX^TM green; (D): cells treated with compound 10 using PI; (E cells treated with compound 11 using SYTOX^TM green; (F): cells treated with compound 11 using PI. The figure with standard deviation is available in Supplementary Materials.

Figure 8

Images of C. neoformans treated with varied concentrations of compound 11. (A1,A2) Cells treated with only SYTOX^TM green; (B1,B2) cells treated with Triton X-100 and SYTOX^TM green; (C1,C2) cells treated with 0.5×MIC of 11 and SYTOX^TM green; (D1,D2) cells treated with 1×MIC of 11 and SYTOX^TM green; (E1,E2) cells treated with 2×MIC of 11 and SYTOX^TM green; (F1,F2) cells treated with 4×MIC of 11 and SYTOX^TM green; (G1,G2) cells treated with 8×MIC of 11 and SYTOX^TM green; (H1,H2) cells treated with only PI; (I1,I2) cells treated with Triton X-100 and PI; (J1,J2) cells treated with 0.5×MIC of 11 and PI; (K1,K2) cells treated with 1×MIC of 11 and PI; (L1,L2) cells treated with 2×MIC of 11 and PI; (M1,M2) cells treated with 4×MIC of 11 and PI; (N1,N2) cells treated with 8×MIC of 11 and PI.

Figure 9

Reactive oxygen species (ROS) study for 4″,6″-disubstituted AKs and compound 14 structure.