Abstract
Background
The rising prevalence of fungal infections and challenges such as adverse effects and resistance against existing antifungal agents have driven the exploration of new antifungal substances.
Methods
We specifically investigated naphthoquinones, known for their broad biological activities and promising antifungal capabilities. It specifically examined the effects of a particular naphthoquinone on the cellular components of Candida albicans ATCC 60193. The study also assessed cytotoxicity in MRC-5 cells, Artemia salina, and the seeds of tomatoes and arugula.
Results
Among four tested naphthoquinones, 2,3-DBNQ (2,3-dibromonaphthalene-1,4-dione) was identified as highly effective, showing potent antifungal activity at concentrations between 1.56 and 6.25 μg mL−1. However, its cytotoxicity in MRC-5 cells (IC50 = 15.44 µM), complete mortality in A. salina at 50 μg mL−1, and significant seed germination inhibition suggest limitations for its clinical use.
Conclusions
The findings indicate that primary antifungal mechanism of 2,3-DBNQ might involve disrupting fungal membrane permeability, which leads to increased nucleotide leakage. This insight underscores the need for further research to enhance the selectivity and safety of naphthoquinones for potential therapeutic applications.
Introduction
Fungi with pathogenic potential for humans have been increasingly recognized as etiologically relevant for severe infections in a growing number of both immunocompromised and immunocompetent patients in recent years. Primarily, this increase has been attributed to a rise in patients with acquired immunosuppression due to agents like the human immunodeficiency virus (HIV) and in those who develop neutropenia in response to chemotherapy [1]. Although antifungal resistance is already a well-recognized issue of concern, most attention and public resources remain focused on researching and developing new drugs against multidrug-resistant bacteria [2].
Nevertheless, the global emergence of multidrug-resistant fungi, such as certain lineages of Candida auris, has heightened alertness in clinical settings [3]. In particular, the ability of C. auris to spread rapidly among ill patients and in critical care units, as well as its propensity to develop resistance to the main classes of antifungal agents, including azoles (i.e., fluconazole), polyenes (amphotericin B), and echinocandins, makes its clinical management challenging and calls for strict infection control measures upon its detection [4–6].
Naphthoquinones are molecules in the quinone class with two aromatic rings forming their chemical backbone. They are synthesized by various plant families (Bignoniaceae, Ebenaceae, Droseraceae, Juglandaceae, Plumbaginaceae, Boraginaceae, among others) and can also be found as secondary metabolites in various algae, fungi, bacteria, and even some animals [7]. These molecules exhibit significant biological activities, such as antibacterial, antiviral, antioxidant, antiparasitic, cytotoxic, and antifungal properties [8]. The antifungal potential of semi-synthetic naphthoquinones was evaluated against 89 fungal isolates, and a compound named IVS320 was shown to be particularly promising. Specifically, it demonstrated best minimum inhibitory concentration (MIC) values for all tested cultures, primarily for Candida species and dermatophytes [9].
This study aimed to evaluate the antifungal potential of four naphthoquinones against fungal reference isolates (Candida spp., Sporothrix spp., Trichophyton spp., and Fusarium spp.), to determine their cytotoxic profile in MRC-5 human fibroblast cells, to assess toxicity using Artemia salina, and to examine phytotoxicity in tomato (Solanum lycopersicum) and arugula (Eruca sativa) seeds. Additionally, the study investigated the mechanism of action of the naphthoquinone derivate with the best antifungal efficacy against Candida albicans ATCC 60193.
Materials and methods
Naphthoquinones
In this study, we utilized the following naphthoquinones: lapachol (4-hydroxy-3-(3-methylbut-2-enyl)naphthalene-1,2-dione), 2-methoxynaphthalene-1,4-dione (2-MNQ), 2,3-dibromonaphthalene-1,4-dione (2,3-DBNQ), and 2-chloro-3-(2-fluoroanilino)naphthalene-1,4-dione (2-ClFNQ) as shown in Fig. 1. These compounds were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Stock solutions were prepared at a concentration of 3.2 mg mL−1 and further diluted in RPMI-1640 medium (Roswell Park Memorial Institute) (Sigma-Aldrich, St. Louis, Missouri, USA) to achieve the desired concentrations ranging from 1.56 to 800 μg mL−1 for the assays.
Naphthoquinones derived from 1,4-naphthoquinone investigated in the present study
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00072
Microorganisms
Eleven reference strains from the “Collection of Microorganisms of Medical Interest” at the National Institute for Amazonian Research (INPA - Instituto Nacional de Pesquisas da Amazônia) were used (Table 1). Subcultures were grown in Sabouraud dextrose (KASVI, Madrid, Spain) medium to maintain purity and viability until testing.
Reference microorganisms deposited in the Collection of Microorganisms of Medical Interest of the National Institute for Amazonian Research – INPA
| Microorganisms | Species | Strain designation |
| Yeasts | Candida albicans | ATCC 60193 |
| Candida albicans | ATCC 36323 | |
| Candida krusei | ATCC 34135 | |
| Candida tropicalis | ATCC 13803 | |
| Candida glabrata | ATCC 2001 | |
| Candida parapsilosis | ATCC 22019 | |
| Subcutaneous pathogenic fungi | Sporothrix brasiliensis | CFP 00551 |
| Sporothrix schenckii | CFP 00746 | |
| Dermatophytes | Trichophyton mentagrophytes | ATCC 9533 |
| Trichophyton rubrum | ATCC 28189 | |
| Opportunistic filamentous fungi | Fusarium oxysporum | LM 5643 |
ATCC = American type culture collection.
Antifungal activity assays
The assays applied to determine MIC values were based on the broth microdilution method as outlined in Clinical and Laboratory Standards Institute documents (CLSI) [10, 11]. Briefly, 100 µL of the test substance solutions, diluted in RPMI-1640 (Sigma-Aldrich, St. Louis, Missouri, USA) broth, were dispensed into 96-well microplates. Final concentrations ranged from 1.56 to 800 μg mL−1 for naphthoquinones (NQ), 0.125–64 μg mL−1 for fluconazole (FLU), and 0.0313–16 μg mL−1 for amphotericin B (AmB), both from Sigma-Aldrich (St. Louis, Missouri, USA). An additional, 100 µL of inoculum, containing 2.5 × 10³ cells/mL of yeasts or 2.5 × 10⁴ cells/mL of dermatophytes and opportunistic filamentous agents, were added. The plates were incubated at 35 °C for 24 h for yeasts and for 96 h for dermatophytes and opportunistic filamentous agents. Visual readings were taken after the incubation periods. The MIC was defined as the lowest concentration of naphthoquinones necessary to inhibit 100% of fungal growth compared to the control assay (performed without antifungal substances). After MIC determination, 10 µL aliquots from wells showing no visible fungal growth were inoculated onto Sabouraud dextrose (KASVI, Madrid, Spain) medium to determine the minimum fungicidal concentration (MFC) [12].
Antifungal mechanism of action
Sorbitol protection assay
The MIC of 2,3-DBNQ against C. albicans ATCC 60193 was determined according to CLSI guidelines (1.56–800 μg mL−1), both in the absence and presence of 0.8M sorbitol (Sigma-Aldrich, St. Louis, Missouri, USA), serving as an osmotic stabilizer. MICs were evaluated after 24 h of incubation at 35 °C [9].
Ergosterol effect assay
The MIC of 2,3-DBNQ against C. albicans ATCC 60193 was established following CLSI protocols, similar to previous descriptions, in the absence and presence of various concentrations (200–1600 μg mL−1) of ergosterol (Sigma-Aldrich, St. Louis, Missouri, USA). Amphotericin B (Sigma-Aldrich, St. Louis, Missouri, USA) was used as the reference antifungal drug in this assay. MICs were assessed after 24 h of incubation at 35 °C [9].
Extravasation assay for substances absorbing in the 260 nm spectrum
C. albicans ATCC 60193 cells were cultured under agitation at 35 °C until reaching the early stationary growth phase (18 h of growth) in RPMI medium (Sigma-Aldrich, St. Louis, Missouri, USA). Post-incubation, cells were washed and resuspended in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (0.16M, pH 7.0) (Sigma-Aldrich, St. Louis, Missouri, USA). Microtubes (final volume 1,500 µL) containing the inoculum (3 × 107 cells/mL) and 2,3-DBNQ (at 1× and 4× MIC) were incubated for 2, 4, and 24 h. After the incubation periods, the microtubes were centrifuged at 3,000 g for 5 min using a MiniSpin microcentrifuge (Eppendorf, Hamburg, Germany), and the absorbance of the supernatants (100 µL) was measured at 260 nm using a Gene Quant DNA/RNA spectrophotometer (Eppendorf, Hamburg, Germany). In this assay, 100% extravasation was defined as the absorbance measured with cells treated with SDS (sodium dodecyl sulfate, 2%) (Sigma-Aldrich, St. Louis, Missouri, USA) [13].
Toxicity assays
Due to the significant antifungal potential exhibited by 2,3-DBNQ and 2-MNQ, as detailed in the results section, their toxicity was further evaluated in the microcrustacean A. salina. Additionally, the phytotoxic and cytostatic potential of 2,3-DBNQ was assessed in S. lycopersicum (tomato) and E. sativa (arugula) seeds. All four naphthoquinones were tested in cell viability assays using the MRC-5 human fibroblast cell line.
MRC-5 cell line toxicity assay
This assay followed the protocol established by Ansar Ahmed et al. [14], aiming to analyze cell viability in MRC-5 fibroblast cells after a 24-h exposure to the substance of interest. Cells were seeded in 96-well plates at a density of 0.5 × 104 cells per well. Following a 24-h period for incubation and cell adhesion, they were treated with various concentrations of naphthoquinones (1.56–100 µM). Subsequently, 10 µL of alamarBlue® Cell Viability Reagent (Thermo Fisher Scientific, Waltham, Massachusetts, USA) was added (0.4% stock solution diluted 1:20 in culture medium). After a 3-h period for resazurin metabolization, fluorescence was measured using a microplate reader to quantify cell viability.
Artemia salina toxicity assay
The protocol for this assay followed the guidelines set by Meyer et al. [15]. A. salina cysts were incubated in sterilized synthetic seawater (36 g L−1 marine salt – Ocean Tech Reef Salt, Ocean Technologies Group, London, England) under constant illumination at 28 °C. After 48 h, the hatched nauplii were transferred to 24-well plates. Due to their promising MIC values in the antifungal assay, the naphthoquinones 2,3-DBNQ and 2-MNQ were tested. Stock solutions of the naphthoquinones were prepared at 1 mg mL−1 in 5% DMSO (Merck KGaA, Darmstadt, Germany) and synthetic seawater, then serially diluted (1,000, 500, 300, 100, and 50 μg mL−1) and tested in triplicate. Ten nauplii were used per well, with 5% DMSO and synthetic seawater as controls. After 24 h, survival rates were assessed to determine the LC50 (Lethal Concentration 50), using PoloPlus software (version 1.0, LeOra Software LLC, Parma, MO, USA).
Germination inhibition assay in tomato and arugula seeds
The assay's effectiveness was categorized as follows: moderately active (+) if 0<%I<29%; active (++) if 30<%I<59%; and highly active (+++) if 60<%I<100%.
Statistical analysis
Results were reported as mean ± standard deviation (SD) from three independent experiments, each conducted in triplicate, when necessary. Statistical differences (P < 0.05) in cytotoxicity tests were determined using analysis of variance (ANOVA), followed by Tukey's or Bonferroni's post-tests in GraphPad Prism 6.0 for Windows (GraphPad, San Diego, CA).
Ethics statement
Our study used standard organisms and substances, with all procedures complying with Brazilian regulations and international ethical standards, including the Declaration of Helsinki. No human or animal subjects were involved, so ethical approval was not required.
Results
Antifungal activity
To assess the antifungal potential of naphthoquinones, we determined the MICs of lapachol, 2-MNQ, 2,3-DNBQ, and 2-CIFNQ against 11 well-characterized reference strains of fungi with etiological relevance in human infections. These strains comprised opportunistic yeasts, dermatophytes, subcutaneous pathogenic fungi, and opportunistic filamentous fungi (Table 1). Among the compounds tested, 2-MNQ, 2,3-DNBQ and 2-CIFNQ exhibited antifungal activity, with 2,3-DBNQ showing the most promising effects, particularly against Candida species (MIC ranging from <1.56 to 6.25 μg mL−1) and dermatophytes (MIC <1.56 μg mL−1) (Table 2).
Minimum inhibitory concentrations (MIC) of 100% as measured with the assessed 1–4 naphthoquinone derivatives for well-characterized strains of selected fungal species with etiological relevance for human patients
| Microorganisms | Strain designation | Lapachol | 2-MNQ | 2,3-DNBQ | 2-ClFNQ | AmB | FLU* |
| Minimum Inhibitory Concentration (MIC) µg mL−1 | |||||||
| Candida albicans | ATCC 60193 | >800 | 25 | 3.125 | 200 | 2 | 2 |
| ATCC 36232 | >800 | 12.5 | <1.56 | 100 | 2 | 2 | |
| Candida krusei | ATCC 34135 | >800 | 12.5 | <1.56 | 50 | 4 | 16 |
| Candida tropicalis | ATCC 13803 | >800 | 12.5 | 3.125 | 200 | 4 | 2 |
| Candida glabrata | ATCC 2001 | >800 | 25 | 6.25 | 400 | 4 | 2 |
| Candida parapsilosis | ATCC 22019 | >800 | 6.25 | <1.56 | 100 | 2 | 2 |
| Sporothrix brasiliensis | CFP 00551 | >800 | 50 | 3.125 | 25 | 2 | 64 |
| Sporothrix schenckii | CFP 00746 | >800 | 25 | 3.125 | 12.5 | 8 | 64 |
| Trichophyton mentagrophytes | ATCC 9533 | >800 | 6.25 | <1.56 | 25 | 8 | 16 |
| Trichophyton rubrum | ATCC 28189 | >800 | <1.56 | <1,56 | 50 | 2 | 2 |
| Fusarium oxysporum | LM 5634 | >800 | 50 | <1.56 | 100 | 16 | 32 |
*Minimum inhibitory concentration (MIC) of 50%. AmB: Amphotericin B, FLU: Fluconazole.
So, the results of this study confirmed the considerable antifungal potential of naphthoquinones against fungi that are etiologically relevant to human health, including opportunistic yeasts, dermatophytes, subcutaneous pathogenic fungi, and opportunistic filamentous fungi. In detail, 2,3-DBNQ showed a fungicidal effect in several tested strains, highlighting a promising role of naphthoquinones, especially of 2,3-DBNQ, in the development of new antifungal agents.
Consequently, we further investigated the fungicidal and/or fungistatic properties of the test substance 2,3-DBNQ. Remarkably, 2,3-DBNQ exhibited a fungicidal profile for seven out of the ten tested strains, including Candida krusei, Candida tropicalis, Candida parapsilosis, Sporothrix brasiliensis, Sporothrix schenckii, Trichophyton mentagrophytes, and Fusarium oxysporum. In contrast, 2-MNQ and 2-ClFNQ showed fungicidal activity against four and six strains, respectively.
Mechanisms of action of 2,3-DBNQ
Due to the demonstrated antifungal potential of 2,3-DBNQ, it was selected for further evaluation of its potential mechanisms of biological action. The assessments included assays to examine the interaction of this antifungal compound with the organism's cell wall, cell wall ergosterol, and potential cellular leakage.
The sorbitol protection assay aimed at determining whether 2,3-DBNQ affects the integrity of the fungal cell wall. MIC assessments of 2,3-DBNQ against C. albicans ATCC 60193 were performed in parallel, both in the presence and absence of sorbitol (0.8M), an osmotic protectant used to stabilize fungal protoplasts. The MIC of 2,3-DBNQ remained unchanged in the presence of sorbitol (6.25 μg mL−1) after 24 h of incubation, suggesting that 2,3-DBNQ does not target mechanisms controlling the synthesis or integrity of the fungal cell wall.
To determine whether 2,3-DBNQ affects membrane ergosterol, an ergosterol assay was conducted. This test evaluates whether a compound interacts with ergosterol in the fungal cell membrane by introducing exogenous ergosterol. The results indicated that the MIC of 2,3-DBNQ against C. albicans ATCC 60193 cells did not change in the presence of various concentrations (200–1600 μg mL−1) of exogenous ergosterol, suggesting that 2,3-DBNQ does not significantly interact with ergosterol, a crucial component of the fungal cell membrane (Fig. 2).
Influence of exogenous ergosterol on the MIC (Minimum Inhibitory Concentration) of 2,3-DBNQ (2,3-dibromonaphthalene-1,4-dione) when applied with Candida albicans ATCC 60193.
Axis X represents the two-folding concentrations of exogenous ergosterol in µg mL−1, while axis Y represents how many times the MIC value of the selected naphthoquinone against Candida albicans ATCC 60193 has been altered due to interaction with exogenous ergosterol. Amphotericin B its known for interact with ergosterol, as observed in the graph
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00072
Lastly, we explored whether 2,3-DBNQ induces cellular leakage, resulting in the efflux of nucleotides absorbing at 260 nm from C. albicans ATCC 60193 cells. Concentrations of 2,3-DBNQ at 3.25 μg mL−1 (1× MIC) and 12.5 μg mL−1 led to leakage ranging from 6% to 55.2% after 2, 4, and 24 h (Fig. 3), as compared to the positive control.
Percentage of nucleotide extravasation in Candida albicans ATCC 60193 treated with 1× and 4× MIC (Minimum Inhibitory Concentration) of 2,3-DBNQ (2,3-dibromonaphthalene-1,4-dione). SDS (sodium dodecyl sulfate) 2% was used as positive control.
Axis X represents the time of reading of the experiment in hours, while axis y represents the percentage of cell leakage detected in 260 nm caused by the selected naphthoquinone in Candida albicans ATCC 60193. As observed, when treated with 4× MIC value (25 μg mL−1) cell leakage rises up to 55.6% in 24 h
Citation: European Journal of Microbiology and Immunology 14, 3; 10.1556/1886.2024.00072
Toxicity assays
We selected 2-MNQ and 2,3-DBNQ, identified for their notable antifungal activity, for toxicity evaluations using a human fibroblast cell line (MRC-5), the microcrustacean A. salina, and seeds of S. lycopersicum and E. sativa.
In the toxicity assessment using the human fibroblast cell line (MRC-5), 2-MNQ, 2,3-DBNQ, and 2-ClFNQ exhibited IC50 (Half-maximal inhibitory concentration 50) values of 11.9 µM, 15.4 µM, and 29.2 µM, respectively. Lapachol demonstrated low toxicity, with its IC50 value being undetectable under the experimental conditions.
We also investigated the toxicity of 2-MNQ and 2,3-DBNQ with A. salina. At a concentration of 50 μg mL−1, 2-MNQ caused 63% mortality, while 2,3-DBNQ resulted in 100% mortality. Additionally, we examined the toxicity of 2,3-DBNQ on seeds of S. lycopersicum and E. sativa. At a concentration of 400 μg mL−1, 2,3-DBNQ inhibited S. lycopersicum germination by 64.1%, and at 200 μg mL−1, it caused a 94.1% inhibition of E. sativa germination.
Furthermore, 2,3-DBNQ exhibited dose-dependent lethal effects on A. salina and inhibited the germination of S. lycopersicum and E. sativa seeds. These findings are well in line with the above-mentioned finding that 2-MNQ and 2,3-DBNQ demonstrated moderate cytotoxicity in the human fibroblast cell line.
Discussion
The antifungal properties of naphthoquinones are well documented in the scientific literature. Recent studies [18, 19] demonstrate that these compounds possess a broad spectrum of biological activities, including antifungal, antibacterial, antiviral, and antiparasitic effects [8]. The antifungal efficacy of naphthoquinones is attributed to their redox properties, which facilitate interactions with critical cellular components such as enzymes and fungal DNA [20]. Additionally, the structure-activity relationship of naphthoquinones varies according to the substituents attached to the naphthoquinone ring [21].
Furthermore, previous studies highlight the importance of the 2,3-disubstitution pattern in enhancing antifungal activity [22]. Our findings confirm the potent antifungal efficacy of 2,3-DBNQ and emphasize its potential as a promising candidate for the development of new antifungal agents.
Moving on to the mechanisms of action, the molecular mechanisms of naphthoquinone activity against fungi are not fully understood, but it is believed that they involve multiple targets, including the fungal cell wall, the cellular membrane and intracellular components. Our results indicate that 2,3-DBNQ induces cellular leakage in C. albicans ATCC 60193 cells, suggesting a possible mechanism of action by disrupting membrane permeability, in turn leading to the leakage of intracellular components. This finding is consistent with previous studies [9, 19] suggesting that naphthoquinones can cause oxidative damage due to the production of reactive oxygen species (ROS).
However, one of the main challenges for the clinical use of naphthoquinones is their toxicity, which has been extensively documented in the literature [23, 24]. These compounds have shown various toxic effects, including cytotoxicity, genotoxicity, and mutagenicity [25]. The toxicity of naphthoquinones is associated with their redox properties and their ability to generate ROS, which can cause oxidative damage to cellular components [26].
Despite these challenges, the significant antifungal potential of naphthoquinones, particularly 2,3-DBNQ, provides a solid foundation for further investigations on their applications in treating fungal infections. Future research should focus on elucidating the exact molecular mechanisms underlying the cellular leakage induced by 2,3-DBNQ, as well as on identifying potential synergistic interactions with existing antifungal agents. Additionally, evaluating the efficacy of naphthoquinones in in-vivo infection models and exploring their safety profile in animal studies will be essential steps toward developing novel antifungal therapeutics based on these compounds.
Conclusions
In summary, this study highlights the considerable antifungal properties of naphthoquinones, with 2,3-DBNQ showing the most notable activity against fungi relevant to human patients, including Candida species and dermatophytes. Our findings contribute to the growing body of evidence supporting the potential of naphthoquinone-based antifungal agents, given their broad biological activities and diverse potential modes of action. Further research into the precise molecular mechanisms and optimal applications of these compounds is necessary, as they could offer a valuable addition to the arsenal of antifungal therapies, addressing the persistent challenge of drug-resistant fungal infections.
Funding sources
This work was supported by the Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) for the funding of the research (EDITAL N. 010/2021- CT&I ÁREAS PRIORITÁRIAS and EDITAL N. 006/2019 – UNIVERSAL AMAZONAS and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) (Finance code 001).
Authors' Contributions
Contributions to the study were as follows: JDRA – study concept and design, investigation, analysis and interpretation of data, manuscript writing, statistical analysis; RSKF – validation of data, statistical analysis; NSOS – writing – review and editing; ACAC – validation of data; ESL – investigation; JGSO – investigation, writing – review and editing; JVBS – study concept and design, investigation, analysis and interpretation of data, manuscript writing, supervision, funding acquisition, approval of final version; ESS – writing – review and editing, formal analysis, supervision, approval of final version; HF – writing – review and editing, approval of final version.
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be interpreted as a potential conflict of interest.
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