Antifungal activity of synthetic naphthoquinones against dermatophytes and opportunistic fungi: preliminary mechanism-of-action tests
© Ferreira et al.; licensee BioMed Central Ltd. 2014
Received: 19 March 2014
Accepted: 17 June 2014
Published: 6 July 2014
This study evaluated the antifungal activities of synthetic naphthoquinones against opportunistic and dermatophytic fungi and their preliminary mechanisms of action. The minimum inhibitory concentrations (MICs) of four synthetic naphthoquinones for 89 microorganisms, including opportunistic yeast agents, dermatophytes and opportunistic filamentous fungi, were determined. The compound that exhibited the best activity was assessed for its action against the cell wall (sorbitol test), for interference associated with ergosterol interaction, for osmotic balance (K+ efflux) and for membrane leakage of substances that absorb at the wavelength of 260 nm. All tested naphthoquinones exhibited antifungal activity, and compound IVS320 (3a,10b-dihydro-1H-cyclopenta [b] naphtho [2,3-d] furan-5,10-dione)-dione) demonstrated the lowest MICs across the tested species. The MIC of IVS320 was particularly low for dermatophytes (values ranging from 5–28 μg/mL) and Cryptococcus spp. (3–5 μg/mL). In preliminary mechanism-of-action tests, IVS320 did not alter the fungal cell wall but did cause problems in terms of cell membrane permeability (efflux of K+ and leakage of substances that absorb at 260 nm). This last effect was unrelated to ergosterol interactions with the membrane.
The recent high incidence of fungal infections is due to the increased number of immunocompromised patients who are infected with HIV or have undergone organ transplantation or chemotherapy . Although effective antifungal agents are currently available, side effects such as toxicity, drug interactions, inadequate pharmacokinetic properties and the development of resistance have been reported . Therefore, new active entities that are safer, more potent and broad spectrum are highly desired as antifungal agents.
Among natural substances, naphthoquinones (found in bacteria, fungi, animals and plants) have attracted interest in recent years due to both their importance in vital biochemical processes and their several known biological activities, such as antitumor, antibacterial and antiviral . Indeed, a series of studies have demonstrated that naphthoquinone derivatives exhibit antifungal activity [4, 5]. Additionally, the antifungal activity of four synthesized compounds was reported against six species of Candida isolated from the oral cavities of patients with removable prostheses .
In this work, the antifungal activities of the compounds 2,2-dimethyl-2,3-dihydronaphtho [2,3-b] furan-4,9-dione, 2,2-dimethyl-2,3-dihydronaphtho [1,2-b] furan-4,5-dione, 3a,10b-dihydro-1H-cyclopenta [b] naphtho [2,3-d] furan-5,10-dione)-dione and 7,9a-dihydro-6bH-cyclopenta [b] naphtho [2,1-d] furan-5,6-dione were evaluated against 89 fungal cultures, including 29 opportunistic yeasts, 40 filamentous fungi and 20 opportunistic dermatophytes. The mechanism of action of the compound with the most effective antifungal activity was investigated using Candida albicans strain ATCC 36232.
Microorganisms belonging to the INPA collection and from ATCC used in bioassays
Group of organisms
Lines of code
Candida albicans (n = 1)
Candida albicans (n = 10)
56/04; 377/06; 839/10; 113/01; 07/10; 143/05; 62/08; 100/04; 322/05; E2/09
Candida parapsilosis (n = 09)
U.1068; U.968/07; U.784/78; U.1047/08; 248/95; U1059/08; U864/07; U840; U1018/08
Cryptococcus neoformans (n = 5)
12/98; WM 148/10; WM626/10; WM628/10; WM629/10
Cryptococcus gattii (n = 4)
WM 161/10; WM178/10; WM179/10; WM 779/10
Microsporum canis (n = 10)
93; Tcapt 178; D42; 794; 233; PV 78; Tcapt 209; 274; 88; Tcapt 215/03
Trichophyton rubrum (n = 10)
U80/99; U548; U656; U1185; U819; U1077; U855; Tp13; Tp269; Tcorp18
Trichophyton tonsurans (n = 10)
T. capt 300/08; U763/06; U763/06; U654/06; T corp 327/03; U1113/09; T corp 07; U68; U104; Tp 10 Tp42
Microsporum gypseum (n = 10)
PV 66/99; U1197/08; 680/07; 91/94; Tcorp 401/07; Tcorp 8/10; PV 56/99; Tcorp 494/08; T corp 282/02; Tcorp 467/08
Opportunistic filamentous fungi
Aspergillus spp (n = 10)
U72; U841; U896; U918; U1124; Tp465; PV17; P640; PV672; P218
Fusarium spp (n = 10)
U1048/08; U37/10; U57/10; U998/08; U1028/08; U500/04; U1012/08; U557/04; U1128/09; U1118
Antifungal activity assay
Minimum inhibitory concentration (MIC) assays were performed with the broth microdilution method, as described by the CLSI (Clinical and Laboratory Standards Institute) in documents M27-A2/CLSI  and M38-A . Briefly, 100 μL of each evaluated compound diluted in RPMI 1640 broth was added to 96-well microplates, with the final concentrations of the compounds ranging from 100 to 0.2 μg/mL (naphthoquinones) and from 64 to 0.06 μg/mL (ketoconazole). Next, 100 μL of an inoculum containing 2.5 × 103 cells/mL of opportunistic yeasts, 2.5 × 105 cells/mL of dermatophytosis agents or 2.5 × 104 cells/mL of opportunistic filamentous fungi was added to the microplate. The microdilution plates were incubated at room temperature (35°C) for 24 to 48 hours (opportunistic yeasts), 15 days (ringworm agents) or 3 days (opportunistic filamentous fungi). Visual readings were performed after 24 hours for the opportunistic yeasts, after 48 hours for the opportunistic filamentous fungi and after 120 hours for the dermatophytes. The MIC was defined as the lowest concentration of the compound causing 50% inhibition of microorganism growth compared to the control without inhibitors.
The antifungal mechanism of action of naphthoquinone IVS320 (3a,10b-dihydro-1H-cyclopenta [b] naphtho [2,3-d] furan-5,10-dione)-dione) was evaluated using the yeast C. albicans strain ATCC 36232 as a model. The influence of IVS320 on the cell wall (sorbitol protection assay) and the effect of ergosterol on the cell membrane (ergosterol effect assay, K+ efflux assay and leakage of substances absorbing at 260 nm) were evaluated.
Sorbitol protection assay
We determined the MIC of IVS320 against C. albicans (ATCC 36232) following CLSI guidelines (from 100 to 0.20 μg/mL) in the presence and absence of 0.8 M sorbitol (Sigma-Aldrich), which acts as an osmotic support. The MICs were determined after 24 hours of incubation at 35°C [9, 10].
Ergosterol effect assay
The MIC of IVS320 for C. albicans (ATCC 36232) was determined following CLSI guidelines in the presence and absence of different ergosterol concentrations (Sigma-Aldrich) (200–800 μg/mL), as previously described [10, 11]. Amphotericin B was used as a control in this assay. The MICs were determined after 24 hours of incubation at 35°C.
Potassium efflux assay
C. albicans (ATCC 36232) was grown for 18 hours at 35°C in RPMI medium. The cells were washed and resuspended to a concentration of 2.5 × 103 cells/mL in deionized water, and 1 mL of this suspension was incubated with IVS320 at the MIC concentration (50 μg/mL) in test tubes at 35°C for various time periods . C. albicans incubated with deionized water only was used as a control. After centrifugation, the amount of K+ released into the supernatant was measured using an atomic absorption spectrophotometer (AA Spectrophotometer 2380, Perkin-Elmer).
Test for leakage of substances absorbing at 260 nm
C. albicans (ATCC 36232) was grown with shaking at 35°C until the early stationary phase (18 hours of growth) in RPMI. After incubation, C. albicans cells were washed and resuspended in MOPS buffer (0.16 M, pH 7.0). Microtubes (final volume 500 μL) containing an inoculum of 5 × 104 cells/mL and 1× or 4× MIC concentrations of IVS320 were incubated for 6 hours. After 1, 2, 4 or 6 hours of incubation, the microtubes were centrifuged (5 min at 3,000 rpm), and the absorbance of the supernatants (100 μL) was measured at 260 nm (Gene Quant DNA/RNA Eppendorf). In this assay, the absorbance due to leakage from cells treated with HClO4 (1.2 M, 100°C, 30 min) was considered 100% [10, 13].
Antifungal activity of new naphthoquinones
Minimum inhibitory concentrations (MICs) of new naphthoquinones against different fungal species
Culture (n = 89)
Minimum Inhibitory Concentration (MIC) μg/mL
C. albicans (n = 10)
6.3 – 100
6.3 – 100
C. parapsilosis (n = 10)
C. neoformans (n = 05)
C. gattii (n = 04)
T. rubrum (n = 10)
M. canis (n = 10)
M. gypseum (n = 10)
T. tonsurans (n = 10)
Fusarium sp. (n = 10)
Aspergillus sp. (n = 10)
Investigation of the mechanism of action of IVS320 against C. albicans ATCC 36232
Action on the cell wall
A sorbitol protection assay was conducted to determine the influence of IVS320 on the integrity of the fungal cell wall. In this assay, MIC determinations for IVS320 against C. albicans ATCC 36232 were carried out in parallel in the presence and absence of sorbitol (0.8 M), which is an osmotic protectant used for the stabilization of fungal protoplasts. If a compound negatively interferes with the fungal cell wall, it will shift the MIC to a higher value in the presence of osmotic support . The MIC of IVS320 did not change in the presence of sorbitol (50 μg/mL) after 72 hours of incubation, which suggests that IVS320 does not act by inhibiting the mechanisms that control cell wall synthesis.
Action on ergosterol
Action on potassium efflux
Leakage of substances absorbing at 260 nm
The 1,4-naphthoquinone structure is common in various natural products and clinically used drugs that are associated with antifungal activity. Previous studies have demonstrated the potential of this class of compounds. Importantly, the work of Sassaki et al.  evaluated the antifungal activities of 1,4-naphthoquinone derivatives and obtained MICs of 8 μg/mL and 16 μg/mL against cultures of Candida albicans and Candida parapsilosis, respectively. Sheng et al. 2011  studied structural changes and the structure/activity of 1,4-naphthoquinone, showing that the introduction of side chains containing sulfur, oxygen or nitrogen atoms at position C2 and/or C3 led to an increase in antifungal activity. These researchers also evaluated the activity of 17 compounds with different side chains and found three compounds that showed broad antifungal activity. One compound, [2-chloro-3-((4-hydroxyphenyl) amino) naphthalene-1,4-dione], presented MICs of 3.12 μg/mL, 1.56 μg/mL and 0.78 μg/mL against Aspergillus fumigatus, C. albicans and C. neoformans, respectively. The antifungal potential of 1,4-naphthoquinone derivatives was also confirmed by Yamashita et al.  and Kategaonkar .
In the present work, all tested naphthoquinones exhibited antifungal activity. These results corroborate those of Freire et al. , who observed the inhibitory activity of these compounds against C. albicans, particularly Nor-α. Nonetheless, these findings require confirmation across a large number of C. albicans isolates as well as other fungal classes. The results of the present study clearly demonstrate the potential of these antifungals against a large number of different isolates of pathogenic species.
IVS320 exhibited the lowest MIC against dermatophytes and Cryptococcus spp. These results are important given that this is the first study evaluating the antifungal action of this compound against these agents. The importance of new drugs for the treatment of dermatophytosis is highlighted by the fact that this disease affects approximately 40% of the world population, that only a small number of drugs are currently available for treatment, that azoles and allylamines have adverse gastrointestinal effects and that there is a high frequency of recurrence [18, 19]. Amphotericin B and flucytosine are widely used for the treatment of cryptococcosis; however, the toxicity of both of these therapeutics is well described, and therapeutic failure is often observed . Due to the robust results for IVS320, this naphthoquinone was selected for assessing its antifungal mechanism of action. A strain of Candida albicans (ATCC 36232) was selected due to the importance of this species in the epidemiology of fungal infections, and the use of an ATCC microorganism will facilitate reproduction of the work reported here .
IVS320 did not alter the structure of the fungal cell wall but did change the permeability of the cell membrane (efflux of K+ and leakage of substances that absorb 260 nm), which was not related to binding with ergosterol. These results are similar to those obtained by Emadi et al. , who evaluated the possible mechanism of action of bis-naphthoquinones and concluded that these compounds cause membrane depolarization.
Accordingly, the toxicity of IVS320 should be extensively evaluated against human cells, even though studies by Freire et al.  showed that this compound exhibited no significant hemolytic activity against mouse erythrocytes and no cytotoxicity against human fibroblasts (NIH 3 T3) at concentrations between 12.5 and 50 μg/mL.
The results of the present study are relevant because they will motivate new in vivo research focusing on the development of new antifungal agents as alternatives for the treatment of ringworm and opportunistic mycoses.
- Borate HB, Sawargave SP, Chavan SP, Chandavarkar MA, Iyer R, Tawte A, Rao D, Deore JV, Kudale AS, Mahajan PS, Kangire GS: Novel hybrids of fluconazole and furanones: design, synthesis and antifungal activity. Bioorg Med Chem Lett 2011, 21: 4873-4878.PubMedView ArticleGoogle Scholar
- Ayati A, Falahati M, Irannejad H, Emami S: Synthesis, in vitro antifungal evaluation and in silico study of 3-azolyl-4-chromanone phenylhydrazones. Daru 2012, 20: 46.PubMedPubMed CentralView ArticleGoogle Scholar
- Ferreira VF, Ferreira SB, da Silva FDC: Strategies for the synthesis of bioactive pyran naphthoquinones. Org Biomol Chem 2010, 8: 4793-4802.PubMedView ArticleGoogle Scholar
- Rahmoun NM, Boucherit-Otmani Z, Boucherit K, Benabdallah M, Villemin D, Choukchou-Braham N: Antibacterial and antifungal activity of lawsone and novel naphthoquinone derivatives. Med Mal Infect 2012, 42: 270-275.PubMedView ArticleGoogle Scholar
- Sheng C, Zhang W: New lead structures in antifungal drug discovery. Curr Med Chem 2011, 18: 733-766.PubMedView ArticleGoogle Scholar
- Freire CPV, Ferreira SB, de Oliveira NSM, Matsuura ABJ, Gama IL, da Silva FDC, de Souza MCBV, Lima ES, Ferreira VF: Synthesis and biological evaluation of substituted α- and β-2,3-dihydrofuran naphthoquinones as potent anticandidal agents. Med Che Comm 2010, 1: 229.View ArticleGoogle Scholar
- National Committee for Clinical Laboratory Standards: Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts. Wayne, PA, USA: Approved Standard M27-A2; 2002.Google Scholar
- National Committee for Clinical Laboratory Standards: Reference Method for Broth Dilution Antifungal Susceptibility Testing of Filamentous Fungi. Wayne, PA, USA: Approved Standard M38-A; 2002.Google Scholar
- Frost DJ, Brandt KD, Cugier D, Goldman R: A whole-cell Candida albicans assay for the detection towards fungal cell wall synthesis and assembly. J Antibiot 1995, 48: 306-310.PubMedView ArticleGoogle Scholar
- Carrasco H, Raimondi M, Svetaz L, Di Liberto M, Rodriguez MV, Espinoza L, Madrid A, Zacchino S: Antifungal activity of eugenol analogues. Influence of different substituents and studies on mechanism of action. Molecules 2012, 17: 1002-1024.PubMedView ArticleGoogle Scholar
- Lunde CS, Kubo I: Effect of polygodial on the mitochondrial ATPase of Saccharomyces cerevisiae. Antimicrob Agents Chemother 2000, 44: 1943-1953.PubMedPubMed CentralView ArticleGoogle Scholar
- Hao G, Shi Y-H, Tang Y-L, Le G-W: The membrane action mechanism of analogs of the antimicrobial peptide Buforin 2. Peptides 2009, 30: 1421-1427.PubMedView ArticleGoogle Scholar
- Tang Y-L, Shi Y-H, Zhao W, Hao G, Le G-W: Insertion mode of a novel anionic antimicrobial peptide MDpep5 (Val-Glu-Ser-Trp-Val) from Chinese traditional edible larvae of housefly and its effect on surface potential of bacterial membrane. J Pharm Biomed Anal 2008, 48: 1187-1194.PubMedView ArticleGoogle Scholar
- Baran M, Borowski E, Mazerski J: Molecular modeling of amphotericin B-ergosterol primary complex in water II. Biophys Chem 2009, 141: 162-168.PubMedView ArticleGoogle Scholar
- Sasaki K, Abe H, Yoshizaki F: In vitro antifungal activity of naphthoquinone derivatives. Biol Pharm Bull 2002, 25: 669-670.PubMedView ArticleGoogle Scholar
- Yamashita M, Kaneko M, Tokuda H, Nishimura K, Kumeda Y, Iida A: Synthesis and evaluation of bioactive naphthoquinones from the Brazilian medicinal plant, Tabebuia avellanedae. Bioorg Med Chem 2009, 17: 6286-6291.PubMedView ArticleGoogle Scholar
- Kategaonkar AH, Pokalwar RU, Sonar SS, Gawali VU, Shingate BB, Shingare MS: Synthesis, in vitro antibacterial and antifungal evaluations of new alpha-hydroxyphosphonate and new alpha-acetoxyphosphonate derivatives of tetrazolo [1, 5-a] quinoline. Eur J Med Chem 2010, 45: 1128-1132.PubMedView ArticleGoogle Scholar
- Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta MS, Gadhwe S: The biology and chemistry of antifungal agents: a review. Bioorg Med Chem 2012, 20: 5678-5698.PubMedView ArticleGoogle Scholar
- Pereira F, Wanderley P: Growth inhibition and morphological alterations of Trichophyton rubrum induced by essential oil from Cymbopogon winterianus Jowitt ex Bor. Braz J 2011, 42: 233-242.Google Scholar
- Posteraro B, Posteraro P, Sanguinetti M: Update on antifungal resistance and its clinical impact. Curr Fungal Infect Rep 2013, 7: 224-234.View ArticleGoogle Scholar
- Sifuentes-Osornio J, Corzo-León DE, Ponce-de-León LA: Epidemiology of invasive fungal infections in Latin America. Curr Fungal Infect Rep 2012, 6: 23-34.PubMedPubMed CentralView ArticleGoogle Scholar
- Emadi A, Ross AE, Cowan KM, Fortenberry YM, Vuica-Ross M: A chemical genetic screen for modulators of asymmetrical 2,2′-dimeric naphthoquinones cytotoxicity in yeast. PLoS One 2010, 5: e10846.PubMedPubMed CentralView ArticleGoogle Scholar
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