Fluoroquinolone compounds have a synergistic antifungal effect with caspofungin against A. fumigatus
Antifungal drugs have various side effects, some of which are fatal in humans. Caspofungin is an antifungal drug of the echinocandin family and inhibits fungal cell wall synthesis by interfering with beta-glucan synthesis. Although the echinocandin family is an effective antifungal drug, it has a paradoxical effect at high concentrations and showed fungistatic function 23. In order to discover the drugs that have a synergistic effect with caspofungin against A. fumigatus, we carried out a drug screening using a chemical library provided by the Korea Chemical Bank (https://chembank.org). The drug screening was performed as shown in Fig. 1. 1x104/ml of A. fumigatus conidia was inoculated to each well containing CM broth media in 96-well plates and 0.5µg/ml of caspofungin was added to each well. And then the eight thousand chemicals were added to each well to a final concentration of 6.2uM and incubated at 37 degrees for three days. From the screening, we were able to find hundreds of compounds with higher activity than caspofungin alone, and then we compared the antifungal activity of those chemicals under two different conditions: with or without caspofungin. Interestingly, 50 compounds showed higher antifungal activity in the presence of caspofungin than alone, and 16 compounds were fluoroquinolone compounds (Table S1)24. We selected one compound (NE-E07) which has the best antifungal activity among 16 compounds for further research.
NE-E07 has a typical fluoroquinolone derivative structure, and to compare the antifungal activity of NE-E07 with other known antifungal drugs, we tested the antifungal activity in the presence of or without caspofungin. As shown in Table 1, we tested representative antifungal drugs from three families: amphotericin B, voriconazole, and caspofungin. They belong to the polyene, azole, and echinocandins classes respectively. The MIC values of amphotericin B, voriconazole, and caspofungin were 6.25µg/ml, 0.84µg/ml, and N/D, respectively, because the cells treated with caspofungin showed residual growth, although growth decreased rapidly. The MIC values of amphotericin B and voriconazole in the presence of caspofungin were the same as for the condition treated alone. This result indicates that amphotericin B and voriconazole do not have a synergistic effect with caspofungin. Interestingly, NE-E07 has a MIC value of 10µg when treated alone, but a MIC value of 0.63µg when treated with caspofungin. And, as shown in Fig. 2A, when NE-E07 was treated with caspofungin, the residual growth of the cells disappeared, which is typical of caspofungin alone. To determine whether this activity of NE-E07 also works against other echinocandin antifungal drugs besides caspofungin or not, we examined its synergistic effect on the activity of the echinocandin antifungal drugs micafungin and anidulafungin, as shown in Fig. 2B. The results showed that both antifungal drugs showed residual growth against A. fumigatus when treated alone, as does caspofungin, but this residual growth is abolished when treated with NE-E07. In addition, the synergistic effect of NE-E07 against to echinocandins is very similar to that of caspofungin. These results suggest that NE-E07 is synergistic with echinocandin-based antifungal drugs.
Furthermore, we confirmed the synergistic effect of NE-E07 using solid media. As shown in Fig. 3, 5x105 conidia were spotted on the plates, which contained 0.5µg/ml of caspofungin and indicated concentration of NE-E07 and incubated for 3 days at 37 degrees. The cells on the plate containing 0.5µg/ml of caspofungin showed slower growth than the cells on the control plate after 3 days. However, no growth was detected when 1.5µg/ml of NE-E07 was added with caspofungin. After 5 days, the cells on the plate containing 0.5µg/ml of caspofungin showed the same growth rate as the cells on the control plate. And we the effect of NE-E07 itself on the growth rate of A. fumigatus was very small. These results indicate that NE-E07 is synergistic with echinocandin-based antifungal drugs.
Fluoroquinolone compounds have no synergistic antifungal effect with amphotericin B and voriconazole against A. fumigatus
To determine whether NE-E07 has synergistic effects with echinocandins and other antifungal drug families, we performed a growth assay in the 96-well plate. As shown in Fig. 4A, cells were treated with the indicated concentrations of amphotericin B and voriconazole, and the MIC values of amphotericin B and voriconazole were 6.2510 µg/ml and 0.8µg/ml, respectively, when treated alone. And the MIC values of amphotericin B and voriconazole were the same when treated with 0.6µg/ml of NE-E07. These results suggest that NE-E07 has no synergistic effect on amphotericin B and voriconazole, and that the synergistic effect of NE-E07 is only for the echinocandins. Conversely, we wanted to know whether amphotericin B and voriconazole have synergistic effects on caspofungin. As shown in Fig. 4B, co-treatment of caspofungin and amphotericin B or voriconazole did not show a synergistic effect on antifungal activity. There was no difference between the growth inhibition of cells treated with voriconazole alone and the growth inhibition of cells treated with voriconazole and caspofungin together. These results suggest that voriconazole does not have a synergistic effect on the activity of caspofungin. The same results were obtained with amphotericin B. Taken together; these results suggest that NE-E07 has a synergistic effect on the echinocandins.
We then investigated the synergistic effects of quinolone antibiotics on caspofungin, as quinolone antibiotics have a similar structure to fluoroquinolone compounds. As shown in Fig. 5, NE-E07, NE-D10, and five quinolone antibiotics were diluted at the indicated concentrations and incubated for three days. NE-E07 showed a synergistic effect with caspofungin as previously shown. NE-D10 is another derivative of NE-E07 and is synergistic with caspofungin. However, all the quinolone antibiotics ciprofloxacin, nalidixic acid, levofloxacin, norfloxacin, and ofloxacin showed no synergistic effect even though they have a similar structure to the NE-E07. These results suggest that the synergistic effect of caspofungin on antifungal activity is only applicable to NE-E07.
Co-administration of caspofungin and NE-E07 increases the survival rate of mice infected with A. fumigatus.
A murine infection assay was performed to investigate the effect of co-administration of caspofungin and NE-E07 in mice. Six-week-old male BALB/c mice were obtained from Doo-Yeol Biotech Co. (Seoul, Korea). Starting on day − 4, mice were injected with cyclophosphamide (150 mg/kg body weight) once every 3 days to suppress their immune system, and hydrocortisone acetate (112.5 mg/kg body weight) was injected on day − 1. Mice were anaesthetized by isoflurane inhalation and treated intranasally with a 20 µl drop of a solution containing 5 x 105 conidia. A drop of the spore solution was placed on the nose of the anaesthetized mice, allowing the mice to inhale the solution without pain. There were 8 mice in each experimental group and two independent experiments were performed. After 24 hours of infection, 1 mg/kg of caspofungin and 5 mg/kg of NE-E07 were administered orally daily, alone, or both, as shown in Fig. 6. When only one drug was administered, 20% hydroxypropyl-β-cyclodextrin (HP-β-CD), the solvent used to dissolve drugs, was administered instead of the drug. As shown in Fig. 6, the mice infected with A. fumigatus started to die 4 days after infection even though NE-E07 was administered, seven mice died after 6 days of infection and 12.5% of the mice survived 8 days after infection. Mice that administered no drug after infection started to die after 6 days, and 0% of the mice survived after 8 days. Mice given caspofungin started to die after 6 days of infection and 12.5% of the mice survived 8 days after infection. Finally, both caspofungin- and NE-E07-treated mice started to die after 6 days and 37.5% of the mice survived 8 days after infection. This result confirmed that NE-E07 has a synergistic effect on the antifungal effect of caspofungin.
NE-E07 may have a similar function to azole antifungal drug
Finally, we sought to identify the mode of action of NE-E07 on A. fumigatus. We performed RNA-seq analysis using the A. fumigatus treated with 1µg/ml NE-E07, 5µg/ml fluconazole, 5µg/ml amphotericin B and 0.5µg/ml caspofungin respectively. Total RNAs were extracted from the drug-treated cells and RNA-seq analysis was performed. As shown in Fig. 7A, transcriptomic analysis was performed and the up-, down- and counter-regulated genes were classified using Venn diagram analysis. Interestingly, the number of commonly regulated genes by NE-E07 treatment was close to that of fluconazole-treated cells among genes with more than a 3-fold change in gene expression. The 35, 2, and 32 genes were co-upregulated, -counter regulated, and -downregulated respectively between the NE-E07 and fluconazole-treated cells. However, 67, 20, and 23 genes were co-upregulated, -counter regulated, and -downregulated, respectively, between the NE-E07 and amphotericin B-treated cells in cells. In addition, 36, 9, and 31 genes were co-upregulated, -counter regulated, and -downregulated respectively between the NE-E07 and caspofungin-treated cells. Relatively fewer genes had changed in gene expression in common with those of NE-E07 and fluconazole-treated cells (Fig. 7B). And a similar regulatory profile was found in the genes regulated by 2-fold changes in gene expression (Figure S1, S2). These results suggest a possible mechanism of action for NE-E07 as an azole antifungal drug.