In Vitro Activity of a Novel Antifungal Compound, MYC-053, against Clinically Significant Antifungal-Resistant Strains of Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis spp.

An urgent need exists for new antifungal compounds to treat fungal infections in immunocompromised patients. The aim of the current study was to investigate the potency of a novel antifungal compound, MYC-053, against the emerging yeast and yeast-like pathogens Candida glabrata, Candida auris, Cryptococcus neoformans, and Pneumocystis species.

of cryptococcosis, a life-threatening infection in immunocompromised hosts (4). Although the rates of cryptococcosis have dropped substantially since the development of highly active antiretroviral therapy, the mortality of HIV patients associated with cryptococcal meningitis remains high. One of the causes of treatment failure is the emergence of azole-resistant and -heteroresistant mutants (21)(22)(23).
Pneumocystis species also affect immunocompromised hosts causing pneumocystis pneumonia (PcP) that, according to the Centers for Disease Control and Prevention, affects over 9% of hospitalized HIV patients in the United States (24) (http://www.cdc.gov/ fungal/diseases/pneumocystis-pneumonia/statistics.html). Pneumocystis spp., originally classified as protozoa, are now classified as fungi but are not susceptible to antifungal drugs, being treated with sulfamethoxazole and pentamidine with a high mortality rate, from 5% to 40% (25,26).

RESULTS
In vitro antifungal activity of MYC-053 against C. glabrata, C. auris, and C. neoformans. The efficacy of MYC-053 against a panel of 20 C. glabrata strains (including seven fluconazole [FLC]-resistant and four caspofungin [CAS]-resistant strains), five C. auris strains, and 18 C. neoformans strains (including 12 FLC-resistant strains) was determined by the broth microdilution method (Table 1). MIC values of MYC-053 for C. glabrata strains varied from 1 to 4 g/ml, while if applying a less restrictive endpoint criterion of 50% inhibitory concentration (IC 50 ), values were in the low g/ml range (0.125 to 0.5 g/ml). C. auris and C. neoformans strains were also sensitive to this compound, with MICs of 1 to 4 g/ml. Notably, the antifungal activity of MYC-053 against the susceptible strains, including the control C. glabrata ATCC 90030 and C. neoformans ATCC 90112 strains, was lower than that of FLC but higher than that of CAS. In contrast, while certain resistant clinical isolates exhibited reduced susceptibility to FLC and CAS, they were highly sensitive to the same concentrations of MYC-053 as the control strains (Table 1).
In vitro antifungal activity of MYC-053 against Pneumocystis carinii and Pneumocystis murina. The responses of Pneumocystis carinii and Pneumocystis murina to MYC-053 were evaluated by a cytotoxicity assay based on ATP-driven bioluminescence (32). The results, expressed as the IC 50 after 24, 48, and 72 h of exposure to the drug, were assigned activity ranks based on the degree of reduction of ATP compared to the untreated controls (33,34) (Table 2). The exposure of P. carinii to 1 g/ml MYC-053 for 72 h resulted in a level of ATP reduction that was slightly lower than that of pentamidine and can be considered moderate activity. However, the increase in MYC-053 concentration to 10 g/ml resulted in smaller amounts of ATP pools than with 1 g/ml pentamidine. The inhibitory effect of MYC-053 against P. murina was higher than that against P. carinii. In this assay, MYC-053 demonstrated activity against P. murina comparable to that with pentamidine, with over 94.4% reduction of the ATP pool following 72 h of exposure, which is considered to indicate marked activity on the efficacy scale (34). Overall, MYC-053 effectively reduced the ATP content of both Pneumocystis species at microgram levels. The following IC 50 values were calculated over 3 days of P. carinii exposure to MYC-053: 3.90 g/ml at 24 h, 2.56 g/ml at 48 h, and 1.61 g/ml at 72 h. Against P. murina, the IC 50 values were 3.30 g/ml at 24 h, 1.50 g/ml at 48 h, and 0.165 g/ml at 72 h.
Activity of MYC-053 against C. glabrata and C. neoformans biofilms. The antibiofilm effects of MYC-053, FLC, and CAS on preformed 48-h-old C. glabrata biofilms and the effects of MYC-053 and FLC on cryptococcal biofilms were evaluated (Table 3). Preformed biofilms were exposed to drugs provided at concentrations equal to 1 to 64    tested against C. neoformans biofilms in this assay, as this microorganism is known to be resistant both in vitro and in vivo to echinocandins.
Importantly, the MIC experiment revealed that MYC-053 exerted a pronounced cidal effect against resistant fungal isolates at concentrations identical to the ones killing susceptible control fungal strains. These data correspond well with the notion that MYC-053 is a representative of a novel chemical class of antifungal agents; it is not relevant to the existing antifungal agents whose use is frequently characterized by cross-resistance (43). Notably, MYC-053 was effective against C. auris, which is often multidrug resistant (44). Although we have only tested the activity of MYC-053 against five C. auris strains, low standard error of the mean (SEM) values in the assay suggested high precision of the measurements, allowing us to determine the mean IC 50 as 1 g/ml and MIC as 4 g/ml. Despite the fact that the MIC values of MYC-053 against C. auris were higher than those against C. glabrata, these values were nonetheless promising given the low susceptibility of certain tested strains to FLC and CAS, with MIC values over 64 g/ml for these antifungals.
MYC-053 was also effective against C. neoformans, with MIC values starting at 1.0 g/ml. These concentrations were dramatically different from the FLC MIC values. Although we did not test the sensitivity of C. neoformans strains against other azoles, it is known that this fungus is commonly cross-resistant to other antifungal agents of this class, including voriconazole (45,46). Therefore, we propose that MYC-053 might be effective against other non-azole-resistant strains of C. neoformans.
This investigation also revealed that MYC-053 was effective against Pneumocystis spp., other yeast-like pathogens that are challenging to treat in immunocompromised patients. The anti-Pneumocystis activity of MYC-053 was promising since, despite being originally classed as protozoa, Pneumocystis spp. are now classified as fungi and continue to be generally treated with antibacterial and antiprotozoan medications (32, Characterization of a Novel Antifungal, MYC-053 Antimicrobial Agents and Chemotherapy 47). The determination of ATP levels for the assessment of MYC-053 activity against Pneumocystis spp. constitutes a highly sensitive assay enabling a reduction in the number of tested organisms (33). The activity of MYC-053 was considered marked and was comparable to the activity of pentamidine against P. murina at the 72-h time point.
To the best of our knowledge, MYC-053 is the first new synthetic compound that can be potentially used against Pneumocystis spp., Candida spp., and Cryptococcus spp. We also revealed that MYC-053 was highly effective against 48-h-old preformed fungal biofilms. At a concentration equal to the MIC, MYC-053 caused a 50% reduction in the viable cell counts in all studied fungal biofilms. The MBEC 90 values of MYC-053 were equal to 1 to 4 times the MIC values. Notably, the MIC/MBEC 50/90 ratios of MYC-053 were significantly lower than those of the control antifungals FLC and CAS. In summary, MYC-053 was equally effective against sessile and planktonic nonresistant organisms and multiresistant clinical isolates.
Taken together, the results of the current study on the efficacy of MYC-053 against certain yeasts and yeast-like pathogens, including ones in a biofilm state, indicate the possibility of developing MYC-053 further into an antifungal drug candidate; however, it requires more in vivo research.
In vitro antifungal susceptibility testing. Microdilution broth susceptibility testing was performed in duplicate according to the CLSI M27-A3 method in RPMI 1640 growth medium (Sigma-Aldrich) to determine the MIC values (48). Standard inoculum for yeast testing was 2.5 ϫ 10 3 CFU/ml. FLC and CAS were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich), whereas MYC-053 was dissolved in sterile water. The IC 50 was defined as the lowest concentration of a drug that at which 50% growth inhibition was observed compared to the growth control. MIC was defined as the lowest concentration of the drug that resulted in no visual growth after 24 h of incubation at 35°C. Fungal isolates were categorized as susceptible, intermediate, or resistant according to the susceptibility breakpoints for antifungals based on CLSI criteria (49)(50)(51)(52). The MIC experiments were performed in triplicate.
In vitro P. carinii and P. murina ATP assays. MYC-053 was diluted directly in the culture medium (0.1, 1, 10, and 50 g/ml). The culture medium was RPMI 1640 containing 20% horse serum, 1% minimum essential medium (MEM)-vitamin solution, 1% MEM-nonessential amino acids (NEAA), 200 U/ml penicillin, and 0.2 mg/ml streptomycin (Sigma-Aldrich). The medium alone and medium containing 10 g/ml ampicillin (Sigma-Aldrich) were the negative controls. Medium supplemented with 1 g/ml pentamidine isethionate was the positive control. Cryopreserved and characterized P. carinii strains isolated from rat lung tissue and P. murina strains isolated from mouse lung tissue were distributed into triplicate wells of 48-well plates (final volume, 500 l; final concentrations, 5 ϫ 10 7 nuclei/ml for P. carinii and 5 ϫ 10 6 nuclei/ml for P. murina). The controls and diluted compounds were added to the cultures and incubated at 35°C under 5% CO 2 . After 24, 48, and 72 h, 10% of the well volume was removed, and ATP content was determined using the ATP-Lite luciferin-luciferase assay (PerkinElmer, Waltham, MA). The ATP-associated luminescence was determined using a spectrophotometer (POLARstar Optima; BMG-Labtech, Germany). Each sample was examined microscopically on the final day of the assay to rule out the presence of bacteria. A quench control assay to determine compound interference in the luciferin/luciferase reaction was negative at all tested concentrations. Background luminescence was subtracted, and triplicate well readings were averaged. For each time point, the percent reduction in ATP content in all groups was calculated as follows: [ATP medium control Ϫ (ATP experimental/ATP medium control)] ϫ 100. The IC 50 was calculated using the INSTAT linear regression program (GraphPad Software, Inc., San Diego, CA). Each test was performed in triplicate.
Effect of MYC-053 on preformed fungal biofilms. A standardized C. glabrata or C. neoformans culture inoculum (200 l; 5 ϫ 10 5 CFU/ml) in RPMI 1640 was added to each well of a 96-well roundbottom polystyrene tissue culture microtiter plate (Sarstedt, Nümbrecht, Germany) (48,53). Following 48 h of incubation at 35°C, biofilm samples were washed twice with phosphate-buffered saline to remove nonadherent cells and then exposed for 24 h to 200 l of RPMI 1640 containing MYC-053, FLC, or CAS at concentrations equal to 1, 2, 4, 8, 16, 32, and 64 times their MICs. Untreated biofilms were used as negative controls. The number of viable fungi in the biofilm was determined by estimating the CFU number. Briefly, to estimate the CFU number, following exposure, well contents were aspirated to prevent antimicrobial carryover, and each well was washed three times with sterile deionized water. Biofilms were scraped thoroughly, with a particular attention to well edges (27). The well contents were aspirated and placed in 2 ml of isotonic phosphate buffer (0.15 M; pH 7.2), the total fungal CFU number was determined by serial dilution and plating on Sabouraud dextrose agar (SDA), and the culture was incubated for 24 h at 35°C. Data were log 10 transformed and were compared with the data for untreated biofilms. The MBEC values of drugs were defined as the concentrations of drug that killed 50% (MBEC 50 ) or 90% (MBEC 90 ) of yeasts in preformed 48-h-old biofilms. All assays included three replicates and were repeated in three independent experiments.
Statistical analysis. The Mann-Whitney U test was used to evaluate the differences between antifungal-treated and control samples. Differences at a P value of Ͻ0.05 were considered significant. The nonparametric paired Wilcoxon signed-rank test was employed to analyze the pre-and postchallenge differences, and a P value of Ͻ0.05 was considered significant.