Journal of Antimicrobial Chemotherapy Advance Access published October 31, 2014
J Antimicrob Chemother doi:10.1093/jac/dku427
Antifungal activity of 6-quinolinyl N-oxide chalcones against Paracoccidioides Nı´vea Pereira de Sa´1, Patrı´cia Silva Cisalpino1, Luciana de Carvalho Tavares2, Leandro Espı´ndola2, Moacir Geraldo Pizzolatti2, Patrı´cia Campi Santos1, Talles Prosperi de Paula1, Carlos Augusto Rosa1, Daniele da Glo´ria de Souza1, Daniel Assis Santos1 and Susana Johann1* Departamento de Microbiologia, Instituto de Cieˆncias Biolo´gicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; 2 Departamento de Quı´mica, Universidade Federal de Santa Catarina, Floriano´polis, SC, Brazil *Corresponding author. Tel: +55-3349-7700; Fax: +55-31-3295-3115; E-mail:
[email protected]
Received 10 June 2014; returned 26 July 2014; revised 25 September 2014; accepted 29 September 2014 Background: Chalcones are an important class of natural compounds that have been widely applied as synthons in synthetic organic chemistry and possess diverse and interesting biological properties. Methods: We conducted tests with the synthetic substances 6-quinolinyl N-oxide chalcones 4c and 4e to determine their antifungal activity against several isolates of Paracoccidioides spp. and their activity in a murine model. We also determined whether the chalcones interacted with other drugs or interfered with the morphology of Paracoccidioides brasiliensis (Pb18) yeast cells. Results: We verified that the substances were active against Paracoccidioides spp., but we did not show an interaction with the drugs tested when only the fractional inhibitory concentration index values were considered individually. We observed that the substances induced in vitro morphological changes. Compounds 4c and 4e showed activity similar to itraconazole in treated mice, as demonstrated by their ability to reduce the number of cfu recovered from the lungs. Histopathological analysis showed that animals treated with 4c presented fewer areas containing inflammatory infiltrate and larger areas of preserved lung tissue, whereas animals treated with itraconazole showed accumulation of inflammatory infiltrate and some granulomas. Mice treated with 4e exhibited inflammation that compromised the tissue. Conclusions: The results presented in this paper confirm the antifungal potential of the chalcones tested. The chalcone 4c was the more effective at controlling the disease in mice and this compound could be a candidate for future studies of the treatment of paracoccidioidomycosis. Keywords: paracoccidioidomycosis, murine model, morphological changes
Introduction Fungi belonging to the genus Paracoccidioides are the agents of paracoccidioidomycosis (PCM). The treatment of PCM is generally protracted (≥1 – 2 years in many cases) and in the absence of therapy, PCM is fatal. Moreover, according to Travassos and Taborda, 1 even after treatment with antifungals there is no assurance of complete clearance of the fungus. This leads to the need for new, safe and effective antifungal compounds for the treatment of PCM. We recently reported that a series of 6-quinolinyl N-oxide chalcones showed interesting antifungal activity against Paracoccidioides brasiliensis (isolate Pb18). The 6-quinolinyl N-oxide chalcones 4c and 4e showed the highest2
antifungal activity and therefore we selected these compounds for the present study.
Materials and methods Experimental substances The N-oxide chalcones 4c and 4e (Table 1) were synthesized in accordance with Tavares et al.2
Fungi and inoculum In this study, we used 14 isolates of P. brasiliensis and 3 isolates of Paracoccidioides lutzii, all of which were members of the collection of
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail:
[email protected]
1 of 5
Downloaded from http://jac.oxfordjournals.org/ at Universidade Federal de Minas Gerais on November 7, 2014
1
de Sa´ et al.
Universidade Federal de Minas Gerais. The fungi were maintained in a chemically defined medium (McVeigh & Morton) and subcultured after 7 days of growth at 378C. The inoculum was prepared as described by Cruz et al.3
Determination of MICs A bioassay of all the Paracoccidioides isolates was performed following the CLSI M27-A3 guidelines4 with modifications suggested by Nakai et al.5 and Johann et al.6 The data are representative of three independent experiments.
Minimum fungicidal concentrations (MFCs)
Time –kill curve procedures A time–kill curve was generated for P. brasiliensis (Pb18). This test was performed in accordance with Klepser et al.8 All of the kill curve experiments were performed in triplicate.
Chequerboard microtitre assay Eight serial 2-fold dilutions of compounds 4c and 4e, amphotericin B, trimethoprim/sulfamethoxazole and itraconazole were prepared in the same way as in the MIC test. A chequerboard pattern was prepared in accordance with Cuenca-Estrella.9
Scanning and transmission electron microscopy Yeast cells of the isolated Pb18 strain were grown in subinhibitory concentrations of compounds 4c and 4e. The cells were prepared as described previously.10 Analyses were performed with a DSM 950 microscope (Zeiss, Germany) and an EM 10 microscope (Zeiss, Germany) in the Center for Acquisition and Image Processing, UFMG, Brazil.
Sorbitol protection assay This assay was performed with compounds 4c and 4e for isolate Pb18 as described previously.11 MICs were read after 10 days at 378C.
Animals Four-to-six-week-old male BALB/c mice were obtained from the biotherium of the Centro de Pesquisas Rene´ Rachou (Belo Horizonte, Brazil). Tests in the animal models were conducted in accordance with the Ethics Committee for Animal Experimentation (CETEA/UFMG), protocol no. 100/2010.
Fungus Yeast cells of the virulent Pb18 strain were cultured in YPD medium at 378C for 7 days. The inoculum was prepared in accordance with Marques et al.12
Animal experiments and treatment A total of 25 mice, divided into groups of 5, were used in each experiment: Groups 1, 2 and 3: animals were infected and treated with compounds 4c, 4e or itraconazole, respectively; Group 4: positive control (infected but not treated); and Group 5: negative control (not infected and treated with PBS). The animals were infected via the intratracheal route as previously described by Santos et al.13 The mice were treated by intraperitoneal
2 of 5
Assay for organ cfu The number of viable microorganisms in the lungs, liver and spleen from the experimental and control mice was determined by counting the cfu in accordance with Maluf et al.14
Histopathology The lungs, liver and spleen were collected, placed into phosphate-buffered formalin and prepared as described previously.12 From the histopathological analysis, a score of the infectious process was built to evaluate the histopathological parameters characteristic of these organs.
Statistical analysis The program GraphPad Prismw version 5 (GraphPad Software) was used for the statistical analysis. The different groups were analysed using analysis of variance and for multiple comparisons the Newman – Keuls test was used. The level of significance used was 0.05.
Results The MIC50 and MIC90 values of compound 4c were 7.8 and 23.4 mg/L, of 4e were 7.8 and 10.4 mg/L, of itraconazole were 0.007 and 0.062 mg/L and of trimethoprim/sulfamethoxazole were 11.7 and 150 mg/L, respectively. The differences in the MICs of the compounds tested, itraconazole and trimethoprim/ sulfamethoxazole were statistically significant (P, 0.05) (Table S1, available as Supplementary data at JAC Online). Regarding the MFC, we found that the fungicidal activity coincided with the MIC or 2× MIC. We found that 4c and 4e were able to reduce by 70% the number of viable cells in the first 100 h of incubation (Figure S1). In tests performed with the Pb18 isolate of the interaction between the experimental substances and amphotericin B, itraconazole and trimethoprim/sulfamethoxazole, no interaction was observed. Scanning and transmission electron microscopy showed that treatment with substance 4c caused flaking of the cell envelope and some cells showed leakage of the cytoplasmic content, retraction of the plasma membrane, several cytoplasmic vacuoles and cytoplasmic clutter. We observed some wilted, burst cells, folds in the plasma membrane, changes in cell morphology and cytoplasmic disorder in cells treated with 4e (Figure S2). No alteration was observed in the value of the MIC in the presence of sorbitol. In the animal model assay, cfu were recovered only from the lungs. Analysis of the lung cfu/g of tissue showed a significant reduction in the number of fungal cells recovered from the animal groups treated with itraconazole or compounds 4c or 4e compared with the positive control (untreated infected group) (P, 0.05) (Figure 1a). Although differences were observed between the three treatments (itraconazole, 4c and 4e), they were not statistically significant. This result points to similarities in the efficiency of
Downloaded from http://jac.oxfordjournals.org/ at Universidade Federal de Minas Gerais on November 7, 2014
The MFC of each compound tested was determined as described by Espinel-Ingroff.7
injection of 4c, 4e and itraconazole for 2 weeks after infection and in all treatments the doses used were 5 mg/kg of body weight per day. This experiment was repeated three times. To investigate potential toxicological effects, uninfected mice were treated with 5 and 15 mg/kg/day 4c and 4e for 15 days. Visual appearance, weight of animals and histopathological tissue from the liver were analysed.
JAC
Chalcones against Paracoccidioides
Table 1. Experimental compounds Substance
Structural formula
4c
Molecular formula
Molecular weight
Log P value
C19H15NO3
305.33
3.18
C18H12FNO2
293.29
3.59
O + N O
OMe
4e
O
Downloaded from http://jac.oxfordjournals.org/ at Universidade Federal de Minas Gerais on November 7, 2014
+ N O
F
(a) 5
*
cfu/g of lung (×104)
4
3
2
1
4e
4c
It
ra
co
Co
na
nt
zo
ro
l
le
0
Liver
Lung
Histopathological score
15
10
* * 5
5
4e
4c
na It
ra
co
fe in tr
ol
co ra It
zo le
ed ct
4e
na
4c
zo le
ed ct in fe ol tr
*
0
0
Co n
10
Co n
Histopathological score
(b) 15
Figure 1. (a) Lung cfu from BALB/c mice infected intratracheally with 1.0×106 yeast cells of the Pb18 isolate of P. brasiliensis and treated with 4c, 4e or itraconazole. The control mice were infected with the same number of yeast cells. Each bar represents the average count of fungi in the lungs and the errors bars indicate SDs. Asterisks denote significant differences (P, 0.05) using the multiple-comparison Newman – Keuls test (n ¼ 15). (b) The histopathological score of infection with P. brasiliensis (Pb18) in the liver and lungs of BALB/c mice treated with 4c, 4e or itraconazole. Asterisks denote significant differences (P,0.05) using the multiple-comparison Newman– Keuls test.
3 of 5
de Sa´ et al.
Discussion This study showed that 4c and 4e are capable of killing fungi in vitro and this activity extends within the genus Paracoccidioides. In yeast treated with compounds 4c and 4e, we found disorder in the plasma membrane and cytoplasm as well as abnormal morphology. Animals treated with 4c and 4e showed a significant reduction in the number of cfu recovered from the lungs when compared with the positive control group that received no treatment. In the animal models, this activity was not significantly different in animals treated with itraconazole, a drug indicated for the treatment of human PCM.15 Marques et al.16 evaluated the potential of the immunizing peptide P10 in BALB/c mice infected with Pb18 and compared this result with treatment with itraconazole (10 mg/kg) and other antifungal drugs. In all tests, immunization with P10 in combination with the other treatments had the best results for controlling infection, with a 60%–80% reduction in lung cfu compared with the control (untreated infected animals). However, P10 immunization or chemotherapy independently achieved a 40%– 60% reduction of the cfu. In the present study, treatment with 4e and 4c (5 mg/kg/day) reduced the cfu by 65% and 75%, respectively, indicating their ability to control the infection. The histopathological analysis and progression score of the disease in mice showed that compound 4c was better able to control inflammation and resolved the infection with better results than treatment with itraconazole and 4e. In summary, our results show great therapeutic potential for compound 4c, owing to its important antifungal activity in a murine model, without granuloma formation and with preservation of lung tissue.
Acknowledgements We are grateful to the Centro de Pesquisas Rene´ Rachou (Belo Horizonte, Brazil) for providing us with the BALB/c mice used in this work.
4 of 5
Funding This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Fundac¸a˜o de Amparo Pesquisa Estado de Minas Gerais (FAPEMIG) and the Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES).
Transparency declarations None to declare.
Supplementary data Table S1 and Figures S1 to S3 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 Travassos LR, Taborda CP. New advances in the development of a vaccine against paracoccidioidomycosis. Front Microbiol 2012; 3: 212. 2 Tavares LC, Johann S, Alves TMA et al. Quinolinyl and quinolinyl N-oxide chalcones: synthesis, antifungal and cytotoxic activities. Eur J Med Chem 2011; 46: 4448– 56. 3 Cruz RC, Werneck SMC, Oliveira CS et al. Influence of different media, incubation times, and temperatures for determining the MICs of seven antifungal agents against Paracoccidioides brasiliensis by microdilution. J Clin Microbiol 2013; 51: 436–43. 4 Clinical and Laboratory Standards Institute. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Third Edition: Approved Standard M27-A3. CLSI, Wayne, PA, USA, 2008. 5 Nakai T, Uno Ikeda F, Tawara S et al. In vitro antifungal activity of micafungin (FK463) against dimorphic fungi: comparison of yeastlike and mycelial forms. Antimicrob Agents Chemother 2003; 47: 1376– 81. 6 Johann S, Sa´ NP, Lima LARS et al. Antifungal activity of schinol and a new biphenyl compound isolated from Schinus terebinthifolius against the pathogenic fungus Paracoccidioides brasiliensis. Ann Clin Microbiol Antimicrob 2010; 9: 30. 7 Espinel-Ingroff A. In vitro fungicidal activities of voriconazole, itraconazole, and amphotericin B against opportunistic moniliaceous and dematiaceous fungi. J Clin Microbiol 2001; 39: 954– 8. 8 Klepser ME, Wolfe EJ, Jones RN et al. Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B tested against Candida albicans. Antimicrob Agents Chemother 1997; 41: 1392 – 5. 9 Cuenca-Estrella M. Combinations of antifungal agents in therapy—what value are they? J Antimicrob Chemother 2004; 54: 854–69. 10 Santos G, Ferri D, Pedro H et al. Oenothein B inhibits the expression of PbFKS1 transcript and induces morphological changes in Paracoccidioides brasiliensis. Med Mycol 2007; 45: 609– 18. 11 Frost DJ, Brandt KD, Cugier D et al. Whole-cell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. J Antibiot (Tokyo) 1995; 48: 306–10. 12 Marques AF, Silva MB, Juliano MAP et al. Additive effect of P10 immunization and chemotherapy in anergic mice challenged intratracheally with virulent yeasts of Paracoccidioides brasiliensis. Microb Infect 2008; 10: 1251– 8.
Downloaded from http://jac.oxfordjournals.org/ at Universidade Federal de Minas Gerais on November 7, 2014
the three treatments under the conditions evaluated. The lungs from infected animals that received no treatment showed multiple pulmonary foci of epithelioid granulomatous inflammation and early pulmonary fibrosis by haematoxylin and eosin staining. The lungs of animals treated with 4c showed a few areas containing inflammatory infiltrate and large areas of preserved lung tissue. The lungs from the group of animals treated with 4e showed excessive infiltration and granulomas. Animals treated with itraconazole exhibited an accumulation of inflammatory infiltrate and some granulomas. In the liver, animals treated with itraconazole and 4c showed no visible morphological changes, but the group treated with N-oxide chalcone 4e showed extensive areas of microsteatosis and hydropic degeneration, similar to untreated animals. The spleen did not show pathological changes (Figure S3). The histopathological score revealed that the inflammatory process of the liver from animals infected but not treated (positive control) was greater than in the groups treated with itraconazole and substance 4c (Figure 1b). The score for compound 4c showed a significant difference compared with untreated animals. Only the lungs of animals that received treatment with 4c showed a pathological score with significant differences in inflammation compared with the control animals. The visual appearance, weight and histopathological tissue from liver of animals treated with both doses of compounds tested were very similar to control group, suggesting a lack of overt toxic effects.
Chalcones against Paracoccidioides
13 Santos PC, Santos DA, Ribeiro LS et al. The pivotal role of 5-lipoxygenasederived LTB4 in controlling pulmonary paracoccidioidomycosis. PLoS Negl Trop Dis 2013; 7: e2390. 14 Maluf MLF, Takahachi G, Svidzinski TIE et al. Antifungal activity of ajoene on experimental murine paracoccidioidomycosis. Rev Iberoam Micol 2008; 25: 163– 6.
JAC 15 Shikanai-Yasuda MA, Filho FQT, Mendes RP et al. Consenso em paracoccidioidomicose. Rev Soc Bras Med Trop 2006; 39: 297–310. 16 Marques AF, Silva MB, Juliano MAP et al. Peptide immunization as an adjuvant to chemotherapy in mice challenged intratracheally with virulent yeast cells of Paracoccidioides brasiliensis. Antimicrob Agents Chemother 2006; 50: 2814– 9.
Downloaded from http://jac.oxfordjournals.org/ at Universidade Federal de Minas Gerais on November 7, 2014
5 of 5
