Synthesis and antifungal activity in vitro of isoniazid Derivatives against histoplasma capsulatum var. Capsulatum

Article abstract of DOI:10.1128/AAC.01654-13

Histoplasmosis is a severe infection that affects millions of patients worldwide and is endemic in the Americas. Amphotericin B (AMB) and itraconazole are highly effective for the treatment of severe and milDer forms of the disease, but AMB is toxic, and the bioavailability of itraconazole is erratic. Therefore, it is important to investigate new classes of drugs for histoplasmosis treatment. In this study, a series of nine isoniazid hydrazone Derivatives were synthesized and evaluated for their antifungal activities in vitro against the dimorphic fungus Histoplasma capsulatum var. capsulatum. The drugs were tested by microdilution in accordance with CLSI guidelines. The compound N=-(1-phenylethylidene)isonicotinohydrazide had the lowest MIC range of all the compounds for the yeast and filamentous forms of H. capsulatum. The in vitro synergy of this compound with AMB against the planktonic and biofilm forms of H. capsulatum cells was assessed by the checkerboard method. The effects of this hydrazone on cellular ergosterol content and membrane integrity were also investigated. The study showed that the compound alone is able to reduce the ergosterol content of planktonic cells and can alter the membrane permeability of the fungus. Furthermore, the compound alone or in combination with AMB showed inhibitory effects against mature biofilms of H. capsulatum. N=-(1-Phenylethylidene)isonicotinohydrazide alone or combined with AMB might be of interest in the management of histoplasmosis. Copyright

Full text of DOI:10.1128/AAC.01654-13

Synthesis and Antifungal Activity In Vitro of Isoniazid Derivatives
against Histoplasma capsulatum var. capsulatum
Rossana de Aguiar Cordeiro,a,b Francisca Jakelyne de Farias Marques,a Rebecca de Aguiar Cordeiro,c Marcos Reinaldo da Silva,d
Angela Donato Maia Malaquias,b Charlline Vládia Silva de Melo,a Jair Mafezoli,d Maria da Conceição Ferreira de Oliveira,d
Raimunda Sâmia Nogueira Brilhante,a,b Marcos Fábio Gadelha Rocha,a,b,e Tereza de Jesus Pinheiro Gomes Bandeira,a,f
José Júlio Costa Sidrima,b
Postgraduate Program in Medical Microbiology, Federal University of Ceará, Fortaleza, Ceará, Brazila; Postgraduate Program in Medical Sciences, Federal University of
Ceará, Fortaleza, Ceará, Brazilb; Faculty of Medicine, Federal University of Ceará, Fortaleza, Ceará, Brazilc; Department of Organic and Inorganic Chemistry, Federal
University of Ceará, Fortaleza, Ceará, Brazild; Graduate Program in Veterinary Science, State University of Ceará, Fortaleza, Ceará, Brazile; Christus College, School of
Medicine, Fortaleza, Ceará, Brazilf
Histoplasmosis is a severe infection that affects millions of patients worldwide and is endemic in the Americas. Amphoteri-
cin B (AMB) and itraconazole are highly effective for the treatment of severe and milder forms of the disease, but AMB is
toxic, and the bioavailability of itraconazole is erratic. Therefore, it is important to investigate new classes of drugs for
histoplasmosis treatment. In this study, a series of nine isoniazid hydrazone derivatives were synthesized and evaluated for
their antifungal activities in vitro against the dimorphic fungus Histoplasma capsulatum var. capsulatum. The drugs were
tested by microdilution in accordance with CLSI guidelines. The compound N=-(1-phenylethylidene)isonicotinohydrazide had
the lowest MIC range of all the compounds for the yeast and filamentous forms of H. capsulatum. The in vitro synergy of this
compound with AMB against the planktonic and biofilm forms of H. capsulatum cells was assessed by the checkerboard method.
The effects of this hydrazone on cellular ergosterol content and membrane integrity were also investigated. The study showed
that the compound alone is able to reduce the ergosterol content of planktonic cells and can alter the membrane permeability of
the fungus. Furthermore, the compound alone or in combination with AMB showed inhibitory effects against mature biofilms of
H. capsulatum. N=-(1-Phenylethylidene)isonicotinohydrazide alone or combined with AMB might be of interest in the manage-
ment of histoplasmosis.
urrently, one of the major drawbacks of treating systemic and evaluate the inhibitory effects of these compounds against H. cap-
C
opportunistic fungal infections is the narrow therapeutic ar- sulatum var. capsulatum. Previous studies have shown the antifun-
gal potential of antituberculosis drugs against the highly virulent
pathogens H. capsulatum var. capsulatum and Coccidioides posa-
dasii (10, 14). We also evaluated the in vitro synergism of one of
these isoniazid analogs, N=-(1-phenylethylidene)isonicotinohy-
drazide [N=-(1-phenyl)], with amphotericin B in both the plank-
tonic and biofilm forms of H. capsulatum var. capsulatum cells.
The effects of this compound alone or in combination with am-
photericin B on cellular ergosterol content and membrane integ-
rity were also investigated.
senal available. In addition, severe side effects, toxicity, primary
resistance of fungal species (1, 2), and therapeutic failures and/or
refractoriness (3) are also considered important issues in the man-
agement of these infections (1, 4). In agents of severe infections,
such as Histoplasma capsulatum (5), Cryptococcus gattii (6), and
Coccidioides species (7), reduced susceptibility to antifungals has
been detected worldwide. Therefore, many studies have been con-
ducted to search for new antifungal drugs (8–10).
Histoplasmosis is a severe infection that affects millions of pa-
tients worldwide and is endemic in the Americas (4, 11). The
disease is caused by H. capsulatum var. capsulatum, a soil-inhab-
iting fungal species that is usually found in association with bird or
bat droppings (11). Respiratory and/or disseminated disease oc-
curs after the inhalation of infectious microconidia, which convert
to a yeast-like form within the lung parenchyma (11).
Nowadays, amphotericin B is the primary choice for induction
therapy in moderate and severe cases of histoplasmosis, while
long-term therapy with itraconazole is recommended for treating
milder infections (11). Although both drugs are considered effi-
cient, some disadvantages can arise during therapy: nephrotoxic-
ity may occur in up to 33% of patients treated with amphotericin
B (12), therapeutic levels of itraconazole may not be attained in
the cerebrospinal fluid of some patients (11), and significantly
reduced blood serum levels of itraconazole have been detected in
HIV patients when this antifungal is coadministered with lopina-
vir and other protease inhibitors (13).
MATERIALS AND METHODS
Microorganisms. A total of 25 strains of H. capsulatum var. capsulatum
isolated in Brazil were included in the study. Most of the strains were
recovered from AIDS patients with disseminated histoplasmosis. Two
strains were isolated from pet cats presenting with cutaneous ulcers de-
rived from disseminated disease. The isolates were identified by the visu-
alization of yeast-like cells inside macrophages on direct Giemsa-stained
smears, as well as by the macro- and microscopic characteristics of cul-
tures in filamentous form. All tested isolates were maintained in potato
Received 30 July 2013 Returned for modification 24 September 2013
Accepted 30 December 2013
Published ahead of print 10 February 2014
Address correspondence to Rossana de Aguiar Cordeiro, rossanacordeiro@ufc.br.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.01654-13
This study aimed to develop isoniazid-derived hydrazones and
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Antifungal Activity of Hydrazones
dextrose agar at room temperature until testing (4). The strains belong to the fungal suspension at concentrations above the MIC onto potato agar
the fungal collection of the Specialized Medical Mycology Center for 15 days at 35°C (10).
(CEMM) of the Federal University of Ceará, Brazil. All procedures were
performed inside a biosafety level 3 laboratory.
The INH-derived compounds were screened for antifungal activity
against randomly chosen H. capsulatum strains (n ϭ 5) in the yeast-like
Synthesis of Schiff bases. The hydrazones were synthesized by a reac- and filamentous forms. Further experimentation was performed only on
tion between isoniazid (INH) and ketones, as described by Ilic´ et al. (15), the drugs that presented the lowest MIC values for both forms. INH de-
with modifications, using the Schiff base method of preparation. The rivatives were tested from 3.9 to 2,000 ␮g/ml, based on H. capsulatum
compounds were prepared with nine different ketones, one by a reaction susceptibility to INH; AMB and ITC were tested from 0.002 to 1.00 ␮g/ml
of acetophenone, 2=-methoxyacetophenone, 3=-methoxyacetophenone, and from 0.0001 to 0.0625 ␮g/ml, respectively (10).
4=-chloroacetophenone, 2=-nitroacetophenone, 3=-methylacetophe-
Synergistic potential of N=-(1-phenylethylidene)isonicotinohydra-
none, 4=-nitroacetophenone, 4=-bromoacetophenone, 2=-phenylace- zide, an isoniazid-derived hydrazone, and AMB by the checkerboard
tophenone (0.003 mol each), and INH (0.006 mol) with 5.0 ml of method. The in vitro synergism between N=-(1-phenylethylidene)isonic-
ethanol. This mixture was sealed in a stainless steel reactor coated with otinohydrazide [N=-(1-phenyl)] and AMB was evaluated in a checker-
Teflon and subjected to microwave radiation for 4 to 5 min. The pu- board assay (19). Checkerboard synergy testing was performed in dupli-
rification process was performed by dissolving the impurities in ethyl cate in microdilution assays to analyze both the yeast and filamentous
alcohol and methyl alcohol at room temperature after successive forms. Combinations were formed with the isoniazid derivative plus AMB
washes, alternating with centrifugation, and the control reactions were at the following concentrations: N=-(1-phenylethylidene)isonicotinohy-
performed by thin-layer chromatography (TLC) to verify the purity of drazide, 23.4 to 187.5 ␮g/ml, and AMB, 0.00098 to 0.00781 ␮g/ml. The
the synthesized compounds. Tests were conducted with silica gel 60 MIC of each drug in combination (MICsyn) was defined as the lowest
(⌽, 63 to 200 ␮m; Carlo Erba, France) in silica 60 F254 plates (Merck, concentration that caused 100% inhibition of visible fungal growth. Drug
Germany) as the stationary phase. The mobile phase was formed by a interactions were classified as synergistic, indifferent, or antagonistic ac-
mixture of acetate-hexane-methanol (7:1:2). The compounds were re- cording to the fractional inhibitory concentration index (FICI). The in-
vealed after exposure to UV at two wavelengths (254 and 365 nm) and teraction was defined as synergistic if the FICI was Յ0.5, indifferent if it
by spraying with vanillin acid solution, followed by heating at 150°C.
All the compounds formed were characterized structurally by high-
was Ͼ0.5 but Ͻ4.0, and antagonistic if it was Ͼ4.0 (20).
Effect of N=-(1-phenylethylidene)isonicotinohydrazide on ergos-
resolution mass spectrometry (HR-MS) with a liquid chromatography terol content of planktonic cells. Total sterols were extracted as described
MS-ion trap-time of flight (LCMS-IT-TOF) spectrometer (Shimadzu) by Moran et al. (21), with modifications. Strains of H. capsulatum in
and by nuclear magnetic resonance (NMR) with a Bruker Avance model yeast-like phase (n ϭ 8) were grown on BHI medium supplemented with
X-ray diffraction (XRD)-500 spectrometer (1H, 300 MHz; 13C, 75 MHz) 10% sheep blood at 35°C for 7 days (16). After this period, a loopful of
using deuterated dimethyl sulfoxide (DMSO-d6) as a solvent. All ketones inoculum (0.5 ϫ 103 to 2.5 ϫ 104 CFU · mlϪ1) was transferred to RPMI
used in this work were obtained from Aldrich and Sigma-Aldrich (Ger- 1640 medium supplemented with N=-(1-phenylethylidene)isonicotino-
many).
hydrazide at 2ϫ MIC, MIC, and sub-MIC (MIC/2) (4). The tubes were
Antifungal susceptibility test of H. capsulatum planktonic cells. incubated for 4 to 5 days at 35°C, and after this period, each cell suspen-
Cultures of H. capsulatum strains (n ϭ 18) in filamentous form were sion was adjusted to a 0.5 McFarland standard to obtain an inoculum of
grown on brain heart infusion (BHI) agar (BD Diagnostics, USA) at 28°C approximately 1.0 to 5.0 ϫ 106 cells/ml, which was then centrifuged at
for 7 days. For yeast-like cells, strains (n ϭ 13) randomly chosen from the 9,660 ϫ g for 3 min. Cellular pellets were suspended in 0.5 ml of alcoholic
set of clinical isolates were grown on BHI medium supplemented with KOH (3.945 g of KOH and 40 ml of sterile distilled water, brought to 100
10% sheep blood at 35°C for 7 days (16, 17). Cultures of H. capsulatum ml with 100% ethanol) and incubated for 1 h at 95°C in a bath. Following
were gently scraped with cotton swabs and transferred to about 4 ml of incubation, the tubes were allowed to cool, and total sterols were extracted
sterile saline solution. The suspensions were homogenized, read at 530 by the addition of 0.6 ml of n-hexane and vigorous vortexing for 5 min.
nm, and adjusted to 90 to 95% transmittance. Afterwards, the suspensions The tubes were centrifuged at 13,416 ϫ g for 1 min, and the entire organic
were diluted to 1:10 with RPMI 1640 medium (Sigma Chemical Co., USA) top layer was transferred to a new tube and mixed with 1 ml of n-hexane.
containing L-glutamine without sodium bicarbonate and buffered to pH Absorbance readings were performed at 295 nm. Ergosterol quantifica-
7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma tion was performed by comparison to the standard curve with ergosterol
Chemical Co., USA) to obtain an inoculum of approximately 0.5 ϫ 103 to (Sigma-Aldrich, Germany). The experiments were performed in dupli-
2.5 ϫ 104 CFU · mlϪ1 (4).
cate. The results were compared with the ergosterol content of cells grown
The INH-derived molecules were weighed, dissolved in dimethyl sul- in RPMI 1640 medium without antifolate drugs, and itraconazole
foxide (DMSO) (Sigma Chemical Co., USA), and further diluted with (MIC/2) was used as a positive control.
RPMI medium to obtain the test concentrations. Stock solutions of am-
Effect of N=-(1-phenylethylidene)isonicotinohydrazide on cell
photericin B (AMB) (Sigma Chemical Co., USA) and itraconazole (ITC) membrane integrity. The effect of N=-(1-phenylethylidene)isonicotino-
(Janssen Pharmaceutica, Belgium) were prepared in DMSO, as suggested hydrazide on cell membrane integrity was performed as suggested by Devi
by CLSI (18). Serial 2-fold dilutions were prepared with RPMI medium et al. (22), with adaptations. Susceptibility testing of N=-(1-phenylethyl-
and stored at Ϫ20°C.
idene)isonicotinohydrazide, INH, and AMB against H. capsulatum in
Susceptibility testing was performed by the broth microdilution pro- yeast-like form was conducted as described above. The cell contents of
tocol (18). Microtiter plates were incubated for 4 to 5 days or 7 days for the wells containing MIC/2 and a positive control without antimicrobials
yeast-like and mold forms, respectively. All isolates were tested in dupli- were harvested by centrifugation at 13,416 ϫ g for 15 min. A volume of the
cate. The controls were grown on RPMI medium buffered with MOPS supernatant was diluted 1:10 in distilled water, and the release of UV-
without antimicrobials. As a quality control for each test, Candida para- absorbing molecules was analyzed by spectrometry at 260 nm for nucleic
psilosis strain ATCC 22019 was also included (18). The MICs of the hy- acids and at 280 nm for proteins.
drazones were based on a previous study with INH and were defined as the
Effect of N=-(1-phenyl) against mature H. capsulatum biofilms. H.
lowest drug concentration that caused 80% inhibition of visible fungal capsulatum biofilms were formed as suggested by Pitangui et al. (23), with
growth (10) For AMB, the MIC was defined as the lowest drug concen- adaptations. Seven-day-old yeast-like cultures were suspended in sterile
tration that prevented any discernible growth (100% inhibition). The saline solution, and the suspensions were adjusted to 2.4 ϫ 106 to 5.5 ϫ
minimum fungicidal concentration (MFC) was defined as the lowest con- 106 cells/ml in BHI broth. Afterward, 0.05 ml of inoculum aliquots was
centration that inhibited fungal growth completely after seeding 0.1 ml of transferred to flat 96-well polystyrene plates and incubated at 35°C for 7 h
May 2014 Volume 58 Number 5
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de Aguiar Cordeiro et al.
FIG 1 Structural formulas of synthetized isoniazid derivatives N=-[1-phenylethylidene]isonicotinohydrazide (A), N=-[1-(2-methoxyphenyl)ethylidene]isoni-
cotinohydrazide (B), N=- [1-(3-methoxyphenyl)ethylidene]isonicotinohydrazide (C), N=- [1-(4-chlorophenyl)ethylidene]isonicotinohydrazide (D), N=- [1-(2-
nitrophenyl)ethylidene]isonicotinohydrazide (E), N=-(1-m-tolylethylidene)isonicotinohydrazide (F), N=- [1-(4-nitrophenyl)ethylidene]isonicotinohydrazide
(G), N=-(1,2-diphenylethylidene)-2-isonicotinohydrazide (H), and N=-[1-(4-bromophenyl)ethylidene]isonicotinohydrazide (I).
in a rotary shaker at 80 rpm for the preadhesion step. Next, the superna- strong inhibitor of fungal biofilms (25). Biofilm viability was moni-
tants were removed and 0.2 ml of BHI broth was added to each well. The tored by the color change of resazurin solution (0.1 mg/ml in RPMI)
plates were further incubated for 72 h as described above, and subse- after incubation at 37°C for Ն6 h (25).
quently, the wells were washed three times with phosphate-buffered saline
Statistical analysis. The antimicrobial susceptibilities were com-
(PBS) solution to remove nonadherent cells. Aliquots of 0.2 ml of BHI pared using one-way analysis of variance (ANOVA) and Tukey’s mul-
medium with antimicrobials were added to viable 72-h biofilms as fol- tiple comparison posttest. Differences between the treatments were
lows: N=-(1-phenyl), 6,250 ␮g/ml to 97.6 ␮g/ml; AMB, 3.12 ␮g/ml to evaluated for significance using the Wilcoxon signed-rank test. A P
0.048 ␮g/ml; and N=-(1-phenyl), 780 ␮g/ml to 11.7 ␮g/ml, plus AMB, value of Ͻ0.05 was considered to be significant. The statistical analyses
1.56 ␮g/ml to 0.023 ␮g/ml. Following incubation at 35°C for 72 h, the were performed with GraphPad Prism 5.0 (GraphPad Software, San
supernatant was aspirated, and an aliquot of 0.1 ml of 0.3% crystal Diego, CA, USA).
violet was added to each well. After 5 min at 25°C, the dye solution was
aspirated and the wells were washed twice with sterile distilled water.
The wells were filled with 0.2 ml of 33% acetic acid, and after 5 min at
RESULTS
Nine isoniazid derivatives were synthesized (Fig. 1), with yields
ranging from 11.2 to 82.1%. The biological activities of the com-
25°C, the mixture was aspirated and read in a spectrophotometer at
550 nm (24). The tests were conducted in duplicate, and the controls
pounds were investigated against strains of H. capsulatum and
phenyl) was compared with that of AMB, which is considered to be a expressed in terms of the MIC in ␮g/ml. Table 1 shows the no-
were grown in medium without antimicrobials. The effect of N=-(1-
TABLE 1 Antifungal activity of synthesized hydrazones against H. capsulatum var. capsulatum (n ϭ 5) in yeast-like and filamentous forms
MIC range (␮g/ml) for:
Compound
Molecular formula
Mol wt
Yeast form
Filamentous form
N=-[1-Phenylethylidene]isonicotinohydrazide
C14H13N3O
C15H15N3O2
C15H15N3O2
C14H12ClN3O
C14H12N4O3
C15H15N3O
C14H12N4O3
C20H17N3O
C14H12BrN3O
239.3
269.3
269.3
273.7
284.3
253.3
284.3
315.4
318.1
7.8–250
62.5–125
31.25–125
125
31.25–125
62.5–250
62.5–250
62.5–250
62.5–125
15.6–250
1,000–2,000
2,000
125–500
250–500
250–500
250–500
1,000–2,000
125–500
N=-[1-(2-Methoxyphenyl)ethylidene]isonicotinohydrazide
N=-[1-(3-Methoxyphenyl)ethylidene]isonicotinohydrazide
N=-[1-(4-Chlorophenyl)ethylidene]isonicotinohydrazide
N=-[1-(2-Nitrophenyl)ethylidene]isonicotinohydrazide
N=-[1-m-Tolylethylidene]isonicotinohydrazide
N=-[1-(4-Nitrophenyl)ethylidene]isonicotinohydrazide
N=-(1,2-Diphenylethylidene)-2-isonicotinohydrazide
N=-[1-(4-Bromophenyl)ethylidene]isonicotinohydrazide
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Antifungal Activity of Hydrazones
TABLE 2 MICs and MFCs of N=-(1-phenylethylidene)isonicotino-
hydrazide against H. capsulatum in yeast-like and filamentous forms
(n ϭ 16)
All of the synthetized molecules were able to inhibit H. capsu-
latum growth in the yeast-like and filamentous forms. However,
some isoniazid-derived compounds were effective only at high
concentrations, as displayed in Table 1. Lower MIC values were
obtained with the isoniazid-derived acetophenone N=-(1-phenyl-
ethylidene)isonicotinohydrazide for both yeast-like and filamen-
tous forms, and for this reason, this drug was chosen for further
experimentation.
The MICs of N=-(1-phenyl) ranged from 7.8 ␮g/ml to 250
␮g/ml and from 31.2 ␮g/ml to 250 ␮g/ml for the yeast-like and
filamentous forms, respectively. The MFC values ranged from
15.6 ␮g/ml to 1,000 ␮g/ml and from 62.5 ␮g/ml to 500 ␮g/ml for
the yeast-like and filamentous forms, respectively (Table 2). No
significant differences between the MIC (P ϭ 0.1808) and MFC
values (P ϭ 0.558) for the phases were detected. The quality con-
trol C. parapsilosis ATCC 22019 showed MIC values in agreement
with CLSI guidelines (26).
The compound N=-(1-phenyl) showed synergistic interactions
with AMB against H. capsulatum in both the yeast-like and fila-
mentous forms. When AMB and N=-(1-phenyl) were combined,
the MICsyn values were approximately 10 times lower than the
MIC values. The hydrazone N=-(1-phenyl) plus AMB had a syn-
ergistic effect against 14 of 18 strains of H. capsulatum in filamen-
tous form, with FICI values that ranged from 0.009 to 0.78 (Table
3). For the yeast-like form, the combination of N=-(1-phenyl) and
AMB showed FICI values that ranged from 0.04 to 0.56. Only
Data (␮g/ml) by form of H. capsulatum
MIC
MFC
Yeast
H. capsulatum
strain
Yeast
Filamentous
Filamentous
03-02-090
03-03-035
03-06-059
05-01-097
05-02-021
05-02-034
05-02-035
05-02-037
05-02-042
05-02-053
05-02-074
05-02-087
05-02-088
05-04-015
05-04-028
05-05-011
125
125
31.2
250
62.5
62.5
125
31.2
62.5
31.2
125
62.5
62.5
125
31.2
62.5
125
500
250
62.5
500
250
62.5
500
62.5
125
250
500
250
250
500
62.5
125
500
15.6
31.2
125
62.5
62.5
7.8
31.2
31.2
62.5
62.5
62.5
250
62.5
31.2
31.2
31.2
62.5
250
500
500
15.6
62.5
62.5
500
500
250
1,000
250
62.5
62.5
menclature, formulas, and molecular weights of the compounds
synthesized. High-resolution mass spectrometry (HR-MS) con-
firmed the molecular weights and empirical formulas of the com-
pounds.
TABLE 3 Synergistic in vitro activity of N=-(1-phenylethylidene)isonicotinohydrazide combined with AMB against H. capsulatum in yeast-like and
filamentous forms (n ϭ 24)
MIC (␮g/ml) for drugs when:
Isolated
Combined
N=-(1-phenyl)a
AMB
Y
N=-(1-phenyl)
AMB
Y
FICI
Y
H. capsulatum
strain
Y
F
F
Y
F
F
F
03-02-090
03-03-035
03-03-054
03-03-055
03-06-059
05-01-096
05-01-097
05-02-018
05-02-021
05-02-034
05-02-035
05-02-042
05-02-053
05-02-074
05-02-084
05-02-085
05-02-086
05-02-087
05-02-088
05-02-091
05-03-040
05-04-015
05-04-028
05-05-011
125
15.6
NTb
62.5
31.2
NT
NT
NT
62.5
62.5
NT
125
31.2
125
NT
0.5
0.5
0.5
0.5
0.5
3.9
3.9
NT
7.8
7.8
NT
NT
NT
3.9
3.9
NT
NT
7.8
7.8
1.95
NT
NT
3.9
7.8
NT
NT
3.9
NT
7.8
3.9
1.0
3.9
0.0078
0.0078
NT
0.0156
0.0156
NT
NT
NT
0.0078
0.0078
NT
0.0019
0.0009
0.0019
NT
0.046
0.265
NT
0.2496
0.3744
NT
NT
NT
0.0702
0.078
NT
0.035
0.034
0.035
NT
NT
0.125
0.125
NT
NT
NT
1.0
NT
NT
0.5
0.25
0.0312
NT
0.5
0.125
0.0312
NT
NT
0.0312
0.5
0.5
1.0
0.5
1.0
0.5
1.0
0.5
NT
NT
NT
46.8
46.8
1.0
NT
1.0
1.0
23.4
NT
NT
23.4
46.8
23.4
3.9
23.4
23.4
1.0
NT
NT
NT
62.5
62.5
31.2
NT
31.2
31.2
31.2
NT
0.0019
0.0019
0.0009
NT
0.0009
0.0009
0.0009
NT
0.753
0.752
0.063
NT
0.034
0.039
0.782
NT
0.5
NT
NT
0.5
0.5
0.5
NT
NT
1.0
NT
NT
NT
62.5
62.5
15.6
NT
0.0156
0.0156
0.0039
NT
0.156
0.156
0.132
NT
NT
NT
NT
62.5
62.5
62.5
62.5
125
125
125
31.2
62.5
NT
0.0009
0.0019
0.0009
0.0019
0.0009
0.0009
0.0009
0.0019
0.0009
NT
0.406
0.753
0.376
0.064
0.189
0.188
0.009
0.126
0.017
NT
NT
NT
NT
62.5
250
NT
NT
62.5
NT
0.00781
0.0156
NT
NT
0.78
0.070
0.062
NT
NT
0.563
NT
0.5
NT
NT
0.0156
NT
0.5
3.9
1.0
NT
NT
0.0156
31.2
0.2808
a Y, yeast-like; F, filamentous.
b NT, not tested.
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de Aguiar Cordeiro et al.
FIG 4 Metabolic activity of mature H. capsulatum biofilms after antimicro-
bial treatments. Yeast-like cells were grown in BHI broth medium for 72 h for
biofilm formation and then treated with different concentrations of N=-(1-
phenyl) or amphotericin B (AMB) alone or in combination. Controls (C) were
formed by incubation in RPMI without antimicrobials. The results are pre-
sented as the percent reduction compared to the controls. The experiments
were conducted in duplicate, and the data are expressed as the means Ϯ SEM
(n ϭ 6). Asterisks indicate statistically significant differences from controls
(P Ͻ 0.05).
FIG 2 Ergosterol content of H. capsulatum. Cells in yeast-like phase were cul-
tured in RPMI medium without antimicrobials as a control (C) or supple-
mented with N=-(1-phenyl) or itraconazole (ITC) at 2ϫ MIC, MIC, and
MIC/2. The experiments were conducted in duplicate, and the data are ex-
pressed as the means Ϯ standard errors of the mean (SEM) (n ϭ 8). The
asterisks indicate statistically significant differences from controls (P Ͻ 0.05).
one strain (H. capsulatum 05-04-015) showed an FICI of Ͼ0.5
(Table 3).
AMB at MIC/2 was not able to induce an outflow of traceable
material from the cells.
Effect of hydrazone N=-(1-phenyl) on ergosterol content of
planktonic cells. The total ergosterol content was determined
for eight strains grown in suprainhibitory (2ϫ MIC), inhibi-
tory (MIC), and subinhibitory (MIC/2) concentrations of the
drug N=-(1-phenyl). A significant reduction in the ergosterol
content was seen when the cells were incubated with N=-(1-
phenyl) at 2ϫ MIC. However, no significant reduction of er-
gosterol content was observed when cells were exposed to in-
hibitory and subinhibitory concentrations of this drug, in
comparison with fungal growth in RPMI medium without an-
timicrobials (Fig. 2). Itraconazole (ITC) was used as a quality
Effect of hydrazone N=-(1-phenyl) against Histoplasma
biofilms. Mature biofilms of H. capsulatum were inhibited by
approximately 50% after treatment with N=-(1-phenyl) at 100ϫ
MIC, 50ϫ MIC, or 25ϫ MIC indistinctly (P Ͻ 0.05). At 1.5ϫ
MIC, the inhibition was approximately 30%. Similar results were
obtained with AMB. Better results were obtained with combina-
tions formed by N=-(1-phenyl) plus AMB, which were able to
reduce the mature biofilms in approximately 80% (Fig. 4).
DISCUSSION
control for the same strains tested, leading to a significant de- Hydrazones are a class of organic compounds with the chemical
crease in the production of ergosterol for the concentrations structure R1R2CANNH2, formed by the replacement of the oxy-
2ϫ MIC, MIC, and MIC/2 (Fig. 2).
gen of the carbonyl group with the functional group -NNH2. The
Effect of N=-(1-phenyl) on cell membrane integrity. azomethine proton (-NHNACH-) formed is an important
Changes in membrane permeability were detected after treat- source for new drug development (9). In recent years, the hydra-
ment of H. capsulatum cells with N=-(1-phenyl) at MIC/2 (Fig. 3). zones have received considerable attention for displaying a variety
The supernatant content of the yeast-like cultures showed absorb- of biological activities, such as antituberculotic, antimicrobial
ing components at 260 nm and 280 nm, suggesting leaking of (27), antitumor (28), analgesic (29), and anti-inflammatory (30)
nucleic acids and proteins, respectively, from the cell. Minor al- properties. Some of these compounds have been described as
terations in cell permeability were also induced by INH at MIC/2. more efficient than standard antibiotics (27).
FIG 3 Absorbance of extracellular content of H. capsulatum cells at 260 nm (A) and 280 nm (B). Cells in yeast-like phase were incubated in RPMI 1640 medium
without antimicrobials as a control (C), N=-(1-phenyl), isoniazid (INH), or amphotericin B (AMB) at MIC/2. The experiments were conducted in duplicate, and
the data are expressed as the mean Ϯ SEM (n ϭ 6). Asterisks indicate statistically significant differences from controls (P Ͻ 0.05).
2508 aac.asm.org
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Antifungal Activity of Hydrazones
Considering the antifungal potential of INH previously de- cell numbers and colorimetric signal may not be linear. In addi-
scribed against the dimorphic pathogens C. posadasii (14) and H. tion, some strains are able to retain the XTT formazan intracellu-
capsulatum (10), in the present study, INH-derived hydrazones larly, which might alter the final measurement. Besides these is-
were evaluated against H. capsulatum in both yeast-like and fila- sues, a particular problem regarding biosafety should be noted
mentous forms. Synthetized hydrazone compounds showed anti- when experiments are performed with biosafety level 3 (BSL3)
fungal activity, although all but one were effective at high con- microorganisms. In general, XTT assays require centrifugation of
centrations only. The compound N=-(1-phenyl) was the most viable cells and further measurement of the absorbance of the
active among all the synthesized derivatives, presenting the supernatant. As centrifugation of living BL3 pathogens must be
lowest MIC values for both yeast-like and filamentous forms, avoided due to the risk of infectious aerosol formation, we have
so we chose it for further experimentation. Given the results, chosen the crystal violet method, since it is an easy and straight-
we investigated the antifungal activity of N=-(1-phenyl) in forward technique, thus bypassing biosafety issues. Biofilm viabil-
combination with the drug of choice, AMB, for the treatment ity was monitored by the color change of resazurin solution (data
of histoplasmosis. The data showed antifungal effects against not shown) in an attempt to corroborate the results obtained by
H. capsulatum planktonic cells, being able to significantly reduce the crystal violet method.
the MIC values of AMB in vitro.
AIDS-related histoplasmosis is in most cases disseminated and
In order to understand the cell target of N=-(1-phenyl), we highly lethal in the absence of an accurate diagnosis and appropri-
investigated its effect on cell ergosterol content and membrane ate antifungal therapy (35). Despite the excellent therapeutic ef-
permeability. According to the results, N=-(1-phenyl) caused a fects of high doses of amphotericin B, studies have shown that
reduction in the cell content of ergosterol in H. capsulatum, mortality rates reach 50% in patients with AIDS and severe histo-
although to a lesser extent than that caused by itraconazole. Visbal plasmosis, usually due to renal insufficiency as a result of aggres-
et al. (31) also evaluated the antiproliferative effects of hydrazone sive use of amphotericin B. Although many patients respond fa-
analogues on the yeast-like phase of Paracoccidioides brasiliensis vorably to treatment, the existence of other comorbidities worsens
and observed growth inhibition of around 90%. The authors the prognosis and can be a life-threatening complication (36). For
found that the hydrazone derivatives affect cell growth by inhib- this reason, the search for new antifungal therapies is increasingly
iting the synthesis of brassicasterol (predominant sterol) and er- necessary.
gosterol in the yeast-like phase. However, one of the compounds
Besides the ability to form biofilms, H. capsulatum utilizes a
did not show an association with any blocking step in the biosyn- wide variety of highly effective virulence mechanisms, including
thesis of sterols, strongly suggesting different modes of action. The temperature-regulated dimorphism and intracellular parasitism.
results here show that both N=-(1-phenyl) and INH are able to The course of the related respiratory disease depends mainly on
alter the membrane permeability of the fungus, making the cells the capacity of the yeast H. capsulatum to survive and replicate
more permeable to nucleic acids and proteins. The dispersion of within alveolar macrophages, the first line of cellular defense
intracellular components suggests that the crucial effect of N=-(1- against H. capsulatum found in the host (23). In a preliminary
phenyl) and INH on H. capsulatum may be the formation of pores experiment (data not shown), we investigated the ability of N=-(1-
in the plasma membrane. Leakage of cytoplasmic material is con- phenyl) to penetrate the macrophages and increase the death rate
sidered to be indicative of serious and irreversible damage to the of the fungus in the phagosome. The results obtained suggest that
cytoplasmic membrane (32). The release of intracellular content is the compound in combination with AMB is capable of restricting
a hallmark of damage and a loss of membrane integrity (22).
the intracellular growth of H. capsulatum, in concentrations 16 to
We also evaluated the antimicrobial potential of hydrazone 64 times smaller than those of N=-(1-phenyl) and AMB, respec-
against mature H. capsulatum biofilms. N=-(1-Phenyl) had con- tively, compared to the MIC of those drugs tested singly. However,
siderable antibiofilm activity, similar to that of AMB. This result further experiments with a greater number of strains should be
reinforces the antibiofilm potential of N=-(1-phenyl), once AMB performed to revalidate the synergistic effect of these drugs against
is considered the most efficient drug against fungal biofilm (24). phagocytosed H. capsulatum cells.
In addition, we found that combination of N=-(1-phenyl) with
The present study shows that N=-(1-phenyl), a hydrazone de-
AMB enhanced the inhibitory activity of the antifungal drug rivative, has antifungal potential against planktonic cells and bio-
against H. capsulatum biofilms, even at lower concentrations. Al- films of H. capsulatum. Further studies should be performed in
though details concerning the structure and dynamics of H. order to evaluate the antifungal and anti-biofilm effects of N=-(1-
capsulatum biofilms in vivo are unknown, we believe that this an- phenyl) in vivo. The results obtained in this study highlight the
timicrobial combination might improve the management of in- importance of finding new molecular targets that can provide
fections caused by H. capsulatum biofilms.
starting points for the rational design of new compounds with
Although there is no standard method for determining the antifungal activity.
inhibitory activity against fungal biofilms, many authors have
chosen colorimetric methods based on metabolic activity mea-
surement, of which the {2,3-bis(2-methoxy-4-nitro-5-sulfophe-
nyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide}
XTT assay is the most commonly used. However, inconsistencies
with this test have already been noted (33, 34). In a seminal study
performed by Kuhn et al. (33) with Candida biofilms, it was clearly
proven that different strains are able to metabolize XTT with dif-
ferent capabilities, making detailed standardization for each strain
necessary. The authors also stated that the relationship between
ACKNOWLEDGMENTS
This study was supported by CNPq (Conselho Nacional de Desenvol-
vimento Científico e Tecnológico), Brazil (grants 302574/2009-3,
PROTAX 562296/2010-7, 141851/2010-4, 552161/2011-0, and
504189/2012-3).
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