3-Selenocyanate-indoles as new agents for the treatment of superficial and mucocutaneous infections

Article abstract of DOI:10.1039/C8NJ04935A

The development of resistance to the current antifungal agents is an alarming problem. Therefore, the search for new molecules capable of combating fungi infections is imperative. This study presents the in vitro activities of a library of seven 3-selenocyanate-indoles against Candida spp. and dermatophytes of the genera Trichophyton and Microsporum. The antifungal susceptibility of compounds 4a and 4b presented geometric mean values of 4.1 and 6.0 μg mL^?1 against Candida spp. and 1.2 and 2.2 μg mL^?1 against dermatophytes following the CLSI guidelines. The 3-selenocyanate-indole 4a showed a fungicidal effect against the whole fungal panel. The toxicological results revealed that the selenocyanates 4a and 4b did not show mutagenicity or cause changes in the human leukocyte cells and were classified as non-irritant by the ex vivo HET-CAM test. The mechanism of action of the 3-selenocyanate-indoles has not been clearly elucidated. However, a genotoxic potential in higher concentrations, observed by the comet assay, leads us to believe that these molecules have their mechanism of action related to the nucleus of the fungal cells.

Full text of DOI:10.1039/C8NJ04935A

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3-Selenocyanate-indoles as new agents for the
treatment of superficial and mucocutaneous
infections†
Cite this: New J. Chem., 2019,
43, 926
Priscilla Maciel Quatrin,a Daiane Flores Dalla Lana,b Luana Candice Genz Bazana,a
Luis Flavio Souza de Oliveira,c Mario Lettieri Teixeira,d Edilma Elaine Silva,e
´
f
William Lopes,
Romulo Faria Santos Canto,g Gustavo Pozza Silveiraae and
ˆ
Alexandre Meneghello Fuentefria ‡*ab
The development of resistance to the current antifungal agents is an alarming problem. Therefore, the
search for new molecules capable of combating fungi infections is imperative. This study presents the
in vitro activities of a library of seven 3-selenocyanate-indoles against Candida spp. and dermatophytes
of the genera Trichophyton and Microsporum. The antifungal susceptibility of compounds 4a and 4b
presented geometric mean values of 4.1 and 6.0 mg mLꢀ1 against Candida spp. and 1.2 and 2.2 mg mLꢀ1
against dermatophytes following the CLSI guidelines. The 3-selenocyanate-indole 4a showed a fungicidal
effect against the whole fungal panel. The toxicological results revealed that the selenocyanates 4a and
4b did not show mutagenicity or cause changes in the human leukocyte cells and were classified as
non-irritant by the ex vivo HET-CAM test. The mechanism of action of the 3-selenocyanate-indoles has
not been clearly elucidated. However, a genotoxic potential in higher concentrations, observed by the
comet assay, leads us to believe that these molecules have their mechanism of action related to the
nucleus of the fungal cells.
Received 2nd October 2018,
Accepted 16th November 2018
DOI: 10.1039/c8nj04935a
rsc.li/njc
available for the treatment of systemic and mucocutaneous
infections caused by Candida spp. The indiscriminate use of these
1. Introduction
Dermatophyte fungi are pathogens capable of invading keratinized drugs has led to resistant and multidrug-resistant infections.4,5
tissues and cause cutaneous infections that are difficult to Currently, C. albicans is the major Candida species in hospital
eradicate. The therapeutic options include ketoconazole, griseo- infections. However, non-albicans Candida infections caused by
fulvin, allylamines, and triazoles. However, resistance to these C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei are
drugs is frequently observed.1–3 Similarly, there are few options becoming quite common. Hence, the development of new
antifungals is imperative.6,7
a
´
˜
´
Programa de Pos-Graduaçao em Microbiologia Agrıcola e do Ambiente,
Privileged scaffolds are structures that can interact with
different molecular cell-targets due to their appropriate molecular
size and shape. Substituents can be designed into these structures,
allowing the preparation of libraries of molecules with great
diversity.8 Indoles are a well-known example of privileged scaf-
folds. Several commercial drugs, which interact with a myriad of
molecular targets, are indole-derivatives, including indomethacin,
ergotamine, frovatriptan, ondansetron, tadalafil, delavirdine,
zafirlukast, and sumatriptan.8
In 1993, 2-((phenylsulfinyl)methyl)-3-(phenylthio)-1H-indole
was identified as a potent HIV reverse transcriptase inhibitor.9
Since then, several libraries of 3-thio-indole derivatives have been
synthesized as new anti-retroviral agents10,11 and as strong poxvirus
inhibitors.12 Also, arylthioindoles, such as 5-bromo-3-((3,4,5-
trimethoxyphenyl)thio)-1H-indole (1) (Fig. 1), have been proposed
as anti-tubulin inhibitors to be used as new antitumor drugs.13,14
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
b
´
˜
ˆ
ˆ
Programa de Pos-Graduaçao em Ciencias Farmaceuticas,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil
c
´
Departamento de Farmacia, Universidade Federal do Pampa, Uruguaiana, Brazil
d
´
´
Laboratorio de Farmacologia, Instituto Federal Catarinense, Campus Concordia,
´
Concordia, Brazil
e
´
ˆ
Departamento de Quımica Organica, Universidade Federal do Rio Grande do Sul,
Porto Alegre, Brazil
f Department of Molecular Biology and Biotechnology,
Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
g
´
˜
ˆ
´
Programa de Pos-Graduaçao em Ciencias da Saude, Universidade Federal de
ˆ
´
Ciencias da Saude de Porto Alegre, Porto Alegre, Brazil
† Electronic supplementary information (ESI) available: Synthesis of 3-selenocyanate-
indoles 4, tables of the mechanism of action, antibacterial susceptibility test, and
HET-CAM assay. See DOI: 10.1039/c8nj04935a
‡ Laboratory of Applied Mycology, School of Pharmacy, Universidade Federal do
Rio Grande do Sul, Porto Alegre, RS, Brazil. E-mail: alexandre.fuentefria@ufrgs.br,
Tel: +55 51 3308 2194
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Fig. 1 Pharmacological properties of 3-chalcogenyl-indoles 1, 2, and 4 as well as the allylic selenocyanate 3.
Commercially available drugs that are first prescribed to treat in human infections.35 Based on these previous studies and
cancer, including 5-fluorouracil, gallium compounds, and mito- with our ongoing program seeking new molecules with anti-
mycin C, are being repurposed as antibacterials.15 With these points microbial properties16,35–37 that can be developed as new leads
in mind, we demonstrate that a library of 3-calcogenyl-indoles 3 for future in vivo studies, we decided to prepare a collection of
(Fig. 1) presents good activities against Staphylococcus aureus seven 3-selenocyanate-indoles 4 (Fig. 1) and screen them
isolates.16 Similarly, anticancer drugs are being repurposed as new against Candida spp. and dermatophytes. Therefore, this work
antifungals. For instance, the geldanamycin derivative 17-AAG, discloses the results of these screening tests and toxicological
which is an Hsp90 inhibitor, dramatically improves the fluconazole studies (cytotoxicity, genotoxicity, mutagenicity, and allergeni-
activity in a Galleria mellonella model of systemic candidiasis.17
city) to the best hit identified. The results are shown in the
Organoselenium compounds have several chemical and following sections.
pharmacological applications18–22 due to their unique mechanisms
of action including broad antimicrobial activity.23 For instance,
diphenyl diselenide and its analogues showed growth-inhibition
2. Results and discussion
and fungistatic activity against filamentous fungi and pathogenic Seven 3-selenocyanate-indoles, 4a–g (Table 1), were synthesized
Candida spp.24 2,20-Dithienyl diselenide demonstrated fungistatic in one chemical step by an electrophilic aromatic substitution
activity toward C. albicans.25 Ebselen analogues presented activity of the respective indole with electrophilic selenocyanate species.
against C. albicans and filamentous fungi.26–28 Selenocyanates have Triselenium dicyanide (TSD), which can be easily prepared
a vast range of biological applications and can be prepared using in situ from malononitrile and selenium dioxide in dimethyl-
different methodologies.29 Efficient preparation of 3-selenocyanate- sulfoxide, was chosen as the electrophilic selenium reagent.32
indoles has been reported in the literature by the use of diverse So, the indoles reacted with TSD in dimethylsulfoxide at room
selenocyanate species.30–34
temperature to yield the target indole selenocyanides in excel-
Recently, a library of allylic-selenocyanates, such as 3, was lent yields (78–99%, ESI†). In a total, five new compounds, 4c–g,
prepared by us as new agents to combat Fusarium spp. involved were prepared.
Table 1 Screening (mg mLꢀ1) of selenocyanate-indoles 4a–g against Candida spp.
R1
R2
R3 R4
C. albicans ATCC 18804 C. tropicalis ATCC750 C. glabrata RL37 C. krusei CK01 C. parapsilosis RL13
4a
4b
4c
4d
4e
4f
4g
FCZ
AFB
H
Br
I
H
H
H
H
CN
H
H
H
H
H
H
H
Ph
H
H
H
H
H
H
6.2
1.5
50
50
450
450
6.2
1.5
3.1
3.1
12.5
50
450
50
3.1
8
0.1
6.2
3.1
25
450
450
450
12.5
8
3.1
6.2
25
25
25
450
450
450
450
12.5
4
CO2CH3
H
H
H
50
Ph CH3 50
6.2
0.5
0.06
1
0.5
0.2
0.03
Fluconazole (FCZ) and amphotericin B (AFB).
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The 3-selenocyanate-indoles 4a–g were screened against a such as CO2CH3 (4d) at the fifth position of the indole ring was
panel of Candida spp.: C. albicans (ATCC18804), C. glabrata deleterious to the activities (Table 1).
(RL37), C. krusei (CK01), C. tropicalis (CT750), and C. parapsilosis
The lead compounds 4a and 4b were screened against a panel
(RL13) (Table 1). The selenocyanate derivatives 4a–g presented of 25 Candida strains and 15 filamentous fungi. These compounds
antifungal activity with MICs r 50 mg mLꢀ1 for C. parapsilosis presented a broad spectrum fungicidal profile against both genera
(RL13). In addition, all compounds were also active against tested and geometric means of 4.1 and 6.0 mg mLꢀ1 toward
C. glabrata (RL37), except for 4e, considering the breakpoint Candida spp. and 1.2 and 2.2 mg mLꢀ1 against Trichophyton and
chosen (50 mg mLꢀ1). Compounds 4a (MICs 3.12–6.25 mg mLꢀ1
)
Microsporum (Table 2). These results are similar to the ones
and 4b (MICs 1.56–6.25 mg mLꢀ1) were active against the whole obtained using FCZ (2.5 mg mLꢀ1), which is the prescribed drug
panel of yeasts and chosen as the lead compounds. Their MICs in the treatment of candidiasis.41 Meanwhile, the selenocyanates
are in the same range as that of the commercial drug fluconazole 4a and 4b were 50–100 times less active than TBF (0.02 mg mLꢀ1
)
(MICs 0.5–8 mg mLꢀ1) (Table 1). Substitution at the 5- (4c and 4d), against dermatophytes. However, since resistance to these micro-
4- (4e), and 3-positions (4f), and the nitrogen of the indole-ring organisms is continuously observed,42–44 4a and 4b might be an
were deleterious to activities against all fungi tested. The only alternative for the reference drug (TBF), especially considering that
exceptions are observed to be 4g against C. glabrata and they do not share the same mechanism of action as that of TBF.
C. parapsilosis, whose MICs are in the same range as those of
4a and 4b to these microorganisms (Table 1).
The MIC of anidulafungin against yeasts and MEC for
filamentous fungi increased significantly in the presence of sorbitol
Next, 3-selenocyanate-indoles 4a and 4b were screened after 7 and 8 days, respectively, as an indication of its fungal cell wall
against a panel of 25 Candida spp. and 15 filamentous fungi. activity. This was not observed for 3-selenocyanate-indoles 4a and 4b,
These compounds demonstrated a broad spectrum of action since their MICs did not change (Table S1, ESI†). Similarly, ergo-
toward yeast (MICs 1.5–12.5 mg mLꢀ1) and filamentous fungi of sterol (100, 150, 200, and 250 mg mLꢀ1) was added into the culture
the genera Microsporum and Trichophyton (0.1–12.5 mg mLꢀ1
(Table 2).
)
medium to verify if the compounds have action on the fungal cell
membrane. Amphotericin B was used as the control. The MICs also
It has been identified that a halogen atom at the 5-position did not vary for the selenocyanate-indoles 4a and 4b (Table S2, ESI†).
of the indole ring of 3-arylthioindoles results in a reduction of The ergosterol and sorbitol assays indicated that the broad-
the free energy associated with the binding of the compounds spectrum antifungal mechanism of action of compounds 4a
to tubulin, which decreases their cytotoxicity.13 It is known that and 4b are not related to interactions with the membrane and
several compounds presenting anti-cancer activity also demonstrate fungal cell wall chemicals. Hence, scanning electron microscopy
antimicrobial capacity.15,17,38–40 Preliminary results of our group (SEM) allowed us to visualize the effects of the 3-selenocyanate-
demonstrated that 3-chalcogenyl-indoles are active toward Gram- indole 4a on the morphology and amount of C. albicans cells
positive bacteria (Fig. 1). The lead compound in these screening after in vitro treatment. A considerable reduction in the number
tests presents a 5-bromoindole moiety.16 Similarly, this work shows of blastoconidia, hyphae, and pseudohyphae was evidenced by
the 3-selenocyanate-indoles 4a and 4b as lead compounds for SEM images after exposure to 4a (Fig. 4). The fungicidal activity
further broad-spectrum antifungal drug development toward of this compound may be related to this effect. The formation of
Candida spp. and dermatophytes of the genera Trichophyton and hyphae and pseudohyphae by yeast is considered an important
Microsporum. These fungi are typical dermatomycosis agents. mechanism of pathogenicity, which facilitates tissue invasion,
Noteworthy, compound 4a also has a 5-bromoindole moiety colonization, and infection of mucous membranes.45,46 There-
as observed in the previous studies with different targets. The fore, this information might be clinically relevant.
presence of iodine (4c) or other electron withdrawing groups
C. albicans (ATCC 18804) was treated with 4a (sub inhibitory
concentration: 3.1 mg mLꢀ1). After 48 h of incubation, the
number of fungal cells and pseudohyphae observed by SEM
reduced (Fig. 2B) in comparison with that observed for the
untreated control (Fig. 2A).
Table 2 Minimum inhibitory concentrations (MIC range/geometric mean,
mg mLꢀ1) of 4a, 4b, fluconazole (FCZ), and terbinafine (TBF)
Yeasts (n = 25)
4a
4b
FCZb
The fungal cells were exposed for 4, 12, 24, and 48 h (C. albicans)
and 8, 12, 24, and 48 h (T. rubrum) to the selenocyanate-indoles 4a
C. tropicalis (n = 5)
C. krusei (n = 5)
C. parapsilosis (n = 5)
C. glabrata (n = 5)
C. albicans (n = 5)
Geometric meana
3.1–6.2
1.5–12.5
3.1
3.1–6.2
1.5–6.2
4.1
1.5–12.5
3.1–12.5
6.2–12.5
3.1–12.5
1.5–6.2
6.0
0.5–16
0.25–32
0.5–2
0.25–32
0.5–32
2.5
Dermatophytes (n = 15)
4a
4b
TBF
M. gypseum (n = 5)
T. mentagrophytes (n = 5)
T. rubrum (n = 5)
0.1–3.1
1.5–6.2
0.4–0.8
1.2
0.7–12.5
0.7–12.5
1.5–3.1
2.2
0.001–0.1
0.008–0.1
0.001–0.06
0.02
Geometric meana
a
b
Fig. 2 SEM of C. albicans strain (ATCC 18804); (A): untreated control.
(B) Treatment with 4a (sub inhibitory concentration, 3.1 mg mLꢀ1).
n = Number of isolates. Geometric mean interspecies. MICs read
after 24 h considering the reduction of fungal growth (D50%).
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(Fig. 3A and C) and 4b (Fig. 3B and D) at concentrations MIC/2, were observed compared to phosphate-buffered saline (PBS,
MIC, MICx2, and MICx4 in a time-kill assay. Compound 4a negative control). Bleomycin (BLE, positive control) reduced the
presented a fungicidal profile (a reduction of 499.9% in log10 cell viability to approximately 20% (Fig. 4A). Similarly, significant
relative to the initial inoculum) against C. albicans (ATCC micronucleus frequency, as well as apoptotic and necrotic
18804) at MICx2 and MICx4 after 4 h of treatment. Meanwhile, processes for compounds 4a and 4b (32 mg mLꢀ1), was not
at MIC/2 and MIC, microbial inhibition was observed after 12 h noticed, inferring a non-mutagenic potential (Fig. 4B). However,
of experiment followed by cell proliferation equivalent to the the comet assay showed that 4a and 4b (32 mg mLꢀ1) caused
positive control (Fig. 3A). A fungicidal effect was also observed significant DNA damage (Fig. 4C).
As disclosed by Pukalskiene et al.,48 the divergence in comet
˙
toward T. rubrum at all concentrations over the initial 8 h of
experiment (Fig. 3C). This complete inhibition of their growth and micronucleus results can be explained by the fact that
in the early hours demonstrates an excellent fungicidal effect proliferating leukocytes were used for the micronucleus assay.
and could be further related to a possible high level of clinical Leukocyte cells have higher repair capacity than cells in the
efficacy. The time-kill assay demonstrated that 4b exhibits a comet’s G0 phase (resting cells). Therefore, the DNA damage
fungistatic effect (a reduction of o99.9% in log10 in relation observed in the comet assay can be repaired during the micro-
to the initial inoculum) against C. albicans (ATCC 18804) at nucleus experiment.49 Since the nuclei of the fungal and
MICx2 and MICx4 after 12 and 24 h of treatment, respectively. mammalian cells have similarities, it is believed that the targets
However, a significant reduction in the activity after 12 to of the compounds are the nucleic acids. Further experiments
24 h of treatment followed by cell proliferation can be observed might be conducted to confirm this hypothesis.
(Fig. 3B). This effect is noted with azole antifungals. These
Most imidazole antifungals present cell toxicity or bioavail-
drugs do not completely eliminate fungal cells but inhibit their ability problems and are formulated for topical use.50 Ketoconazole
growth. The fungistatic mechanism usually works in conjunction is cytotoxic and presents a possible mechanism of mitochondrial
with the immune system of the host, killing pathogenic or dysfunction in liver cells, compromising mitochondrial DNA
opportunistic microorganisms. However, this mechanism of synthesis, resulting in induced apoptosis.51 Thus, even drugs
action might be a major concern in immunocompromised, demonstrating cellular toxicity for systemic use are prescribed
patients leading to a persistent infection.47 Meanwhile, the for topical administration. In addition, in the present study, the
selenocyanate-indole 4b presented a fungicidal profile against toxicity of the selenocyanate-indoles 4a and 4b was evaluated at
T. rubrum (45) at MIC and MICx2 after 24 h and at MICx4 after 8 h of 32 mg mLꢀ1 and the MICs (geometric mean) determined were in the
treatment. Cell proliferation is observed at MIC and MICx2 after 8 h range of 1.2 to 6.0 mg mLꢀ1. Thus, DNA damage is being observed at
of treatment, which would suggest the need for a new therapeutic higher concentrations of the compounds compared to the MICs.
dose. A dose-dependent effect was noted in the first few hours of the
The selenocyanate-indoles 4a and 4b (32 mg mLꢀ1) are found
experiment with T. rubrum, which required a new therapeutic dose to be non-irritant (IS = 2.19 and 3.01, respectively) through the
for complete microbial inhibition, as noticed at MIC and MICx2 HET-CAM assay (Fig. S15, ESI†). The chorioallantoic membrane
concentrations after 24 h of treatment (Fig. 3D).
is highly vascularized and responds to injuries caused by
Selenocyanate-indoles 4a and 4b (32 mg mLꢀ1) were not processes, such as inflammation, similar to that observed in
cytotoxic to human leukocyte cells, since no statistical differences the conjunctival tissue of rabbit eyes.52 This is an indication
Fig. 3 Time-kill assay of selenocyanates 4a (A and C) and 4b (B and D) against C. albicans (ATCC 18804) strain and T. rubrum (45) isolate at: MIC/2(’),
MIC(m), MICx2(.), MICx4(
~
), and control (ꢁ). 4a MIC: 6.25 mg mLꢀ1 (C. albicans ATCC 18804), and 0.78 mg mLꢀ1 (T. rubrum TRU 45); 4b MIC: 1.56 mg mLꢀ1
(C. albicans ATCC 18804), and 1.56 mg mLꢀ1 (T. rubrum TRU 45).
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and ex vivo54 studies. It is assumed that Gram-positive bacteria
adhere or bind to the hyphae of Candida spp. For instance, it has
been shown that C. albicans can transport S. aureus into tissues
disseminating the infection in an oral co-colonization model.
Therefore, co-infection of Candida spp. and S. aureus results in a
more serious infection than that caused by each microorganism
individually.54 3-Selenocyanate-indoles 4a and 4b were also
screened against S. aureus (ATCC 25923) and the MICs determined
were 8 mg mLꢀ1 (see the ESI†), indicating that the compounds
might have antimicrobial capacities, avoiding this kind of
co-infection.
3. Experimental section
3.1. Fungal strains
In total, 40 clinical and ATCC (American Type Culture Collection,
Manassas, VA, USA) strains were used in this study, including
C. tropicalis (ATCC 750, 17P, 72P, RL104, and ATCC 950), C. krusei
(CK01, CK02, CK03, RL102, and DEN43), C. parapsilosis (RL11,
RL13, RL33, RL100, and CP007), C. glabrata (RL03, RL22, RL37,
RL105, and CG09), C. albicans (CA01, CA02, DEB05, DEB09, and
ATCC 18804), M. gypseum (MGY 01, MGY 1, MGY 2, MGY 3, and
MGY 50), T. mentagrophytes (TME 1, TME 2, TME 3, TME 40,
and TME 46), and T. rubrum (TRU 2, TRU 3, TRU 45, TRU 47, and
TRU 51), belonging to the mycology collection of the research
group in Applied Mycology, School of Pharmacy (Universidade
Federal do Rio Grande do Sul, Brazil).
3.2. Synthesis process
3-Selenocyanate-indoles 4 were synthesized in good yields by the
reaction of the respective indole with TSD, which is prepared
in situ from selenium dioxide and malononitrile in DMSO (ESI†).
3.3. Antifungal susceptibility testing
The minimum inhibitory concentrations (MICs) of the seleno-
cyanate-indoles 4 were determined by the broth microdilution
method according to the M27-A3 protocol for Candida spp. and
M38-A2 for filamentous fungi (Microsporum spp. and Trichophyton
spp.).55,56 Serial microdilutions were performed in RPMI 1640
medium, and the experiments were carried out in duplicate.
MICs are defined as the lowest concentration of compounds at
which the microorganisms tested did not demonstrate visible
growth after 48 (Candida spp.) or 96 h (for Microsporum spp. and
Trichophyton spp.). Fluconazole and terbinafine were used as
the reference antifungals against Candida spp. and dermato-
phytes fungi, respectively. The sterility control (negative control:
a drug-free medium) and positive control for fungal cell viability
were used in parallel (ESI†).
Fig. 4 Toxicological analysis to evaluate the cell viability, mutagenicity,
and genotoxicity of selenocyanate-indoles 4a and 4b (32 mg mLꢀ1) toward
human leukocytes. Phosphate-buffered saline (PBS, negative control) and
bleomycin (BLE, positive control).
3.4. Mechanism of action
3.4.1. Ergosterol binding assay and sorbitol protection
that formulations for topical administration might be developed assay. In order to evaluate the ability of selenocyanate-indoles
without further mucous irritation.
4 to form a complex with the sterol of fungal membranes, the
Synergistic effects as a consequence of co-infection of Candida ergosterol assay was performed.57 The exogenous (qualitative)
spp. with Staphylococcus have been demonstrated by in vitro53 ergosterol determination technique was executed with and
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without the addition of ergosterol (Sigma-Aldrich) at concentrations characterized by a decrease Z99.9% in log10 of the CFU mLꢀ1
of 100, 150, 200, and 250 mg mLꢀ1 against C. albicans (ATCC 18804), number compared to the initial inoculum.
C. glabrata (RL37), C. krusei (CK01), C. tropicalis (ATCC 750),
3.6. Toxicity (cell viability, genotoxicity, and mutagenicity assays)
C. parapsilosis (RL13), T. rubrum (TRU45), T. mentagrophytes
(TME46), and M. gypseum (MGY50). Ergosterol was dissolved in The cell culture was prepared using 1 mL of venous blood
dimethylformamide (Sigma-Aldrich) and the solution was added to collected by venipuncture of a young adult volunteer over
RPMI-1640 medium (containing L-glutamine, without sodium 18 years of age without using medication. Lymphocytes (Protocol
bicarbonate, buffered to pH 7.0, Sigma-Aldrich). The microplates approved by the Ethics Committee of the Universidade Federal
were incubated at 35 1C for 48–72 h. The MICs were determined do Pampa under number 27045614.0.0000.5323. The procedure
visually by the absence or presence of fungal growth. Amphotericin was performed with the consent of the volunteer and signing of a
B was used as a control. The assay was performed in duplicate.
term of acceptance of his participation in the research.) obtained
The antimicrobial effect on the integrity of the fungal cell by centrifugation gradient were immediately transferred to the
wall was determined by a sorbitol protection assay.57 The MICs culture medium containing 9 mL of RPMI 1640 medium,
of the compounds were determined with and without addition supplemented with 10% fetal bovine serum and 1% streptomycin/
of sorbitol (0.8 M, Sigma-Aldrich) against C. albicans (ATCC penicillin. Cell culture flasks were stored at 37 1C for 72 h.
18804), C. glabrata (RL37), C. krusei (CK01), C. tropicalis (ATCC The negative control was prepared using 500 mL of PBS buffer
750), C. parapsilosis (RL13), T. rubrum (TRU45), T. mentagrophytes 7.4, and the positive control with 3 mg mLꢀ1 of bleomycin.
(TME46), and M. gypseum (MGY50). Sorbitol was dissolved in the Selenocyanate-indoles 4a and 4b were evaluated at 32 mg mLꢀ1
.
RPMI-1640 culture medium (containing L-glutamine, without The cell viability was determined based on the loss of leukocyte
sodium bicarbonate, buffered at pH 7.0, Sigma-Aldrich). The membrane integrity measured by the trypan blue method. Each
microplates were incubated at 35 1C and the MICs determined group was analyzed in triplicate. About 100 cells were analyzed
were visually by the absence or presence of fungal growth from per slide for each group in a Neubauer chamber at 400ꢂ
the day 2 to the day 7 of incubation for the yeasts and from the magnification.61 The micronucleus test was performed under a
day 4 to the day 8 of incubation for the filamentous fungi. microscope62 (an increase of 1000ꢂ) and 300 cells were counted
Anidulafungin was used as a control. The assay was performed and classified as a number of mononuclear cells with the
in duplicate.
presence of micronucleus. Finally, the comet assay followed
3.4.2. Scanning electron microscopy (SEM). Only one fungal the guidelines for the use of the technique.63–65 In total, 100
strain – C. albicans (ATCC 18804) was selected to perform scanning nucleoids per slide (in triplicate for each group) were selected
electron microscopy (SEM). Selenocyanate-indole 4a was tested at a and analyzed. The nucleoids were classified according to the
concentration of 3.1 mg mLꢀ1. After 48 h of incubation, wells length of the tail for subsequent calculation of the DNA damage
containing the coverslips were washed three times with phosphate index. For classification, the nucleoids received scores from
buffer saline (PBS). After washing, the adhered cells were added 0 (no migration) to 4 (maximum migration).
with 500 mL of glutaraldehyde (2.5%, type 1, Sigma Aldrich),
3.7. Hen’s egg chorioallantoic membrane (HET-CAM) test
diluted with sodium cacodylate (0.1 M, pH 7.2, Sigma Aldrich),
and kept for 1 h at room temperature. Then, the wells were washed Fresh and fertile white eggs (Lohmann selected Leghorn, LSL)
three times with sodium cacodylate (0.1 M, pH 7.2) containing were kept under optimized incubation conditions (38–39 1C,
sucrose (0.2 M) and MgCl2 (2 mM). The adhered cells were 55–60% humidity, 10 days). On the day 10, the eggshell, around
dehydrated by immersion in gradients of acetone solutions. Then, the airspace, was removed using a rotary tool (Dremel, WI).
the samples were subjected to critical point drying (EM CPD 300, Subsequently, 0.3 mL of each selenocyanate-indoles (32 mg mLꢀ1
,
Leica) immediately after dehydration, mounted on metallic 0.5% DMSO solution) and controls (negative control: 0.9% NaCl;
stubs, sputter-coated with a 15–20 nm gold-palladium layer, positive control: 0.1 M NaOH) were added to the CAM of the eggs.
and visualized using a scanning electron microscope (Carl Zeiss Then, the eggs were observed continuously for 5 min for the
EVOs MA10, Oberkochen, Germany) operating at 10 kV.58
appearance of haemorrhage, lysis, and coagulation, which was
documented. In addition, the severity of each reaction after 1 and
5 min was recorded. From this, the irritation score (IS) was
3.5. Time-kill assay
A time-kill assay was carried out with one representative strain calculated using eqn (1) (see the ESI†) The assay was performed
of each genus studied (C. albicans ATCC 18804 and T. rubrum in triplicate. Haemorrhage, lysis, and coagulation time are expressed
45), and the procedures were conducted as previously described in seconds considering the first appearance of blood haemorrhage,
by Klepser et al.59 and Ghannoum et al.,60 for Candida spp. and vessel lysis, and protein coagulation, respectively.66
dermatophytes, respectively. The activity of the selenocyanate-
3.8. Statistical analysis
indoles 4a and 4b was evaluated against the isolates at 0, 4, 12,
24, and 48 h (C. albicans) and 0, 8, 12, 24, and 48 h (T. rubrum) Analysis of variance (ANOVA) followed by the post hoc Bonferroni
at concentrations of MIC/2, MIC, MICx2, and MICx4. Time-kill test was performed in the statistical analysis of the toxicity test
curves were constructed by plotting the log10 CFU mLꢀ1 mean results. Results with P o 0.05 were considered significant. Data
versus the time of exposure of the fungal cells to the selenocyanate- were analyzed using the GraphPad Prism software program (Graph-
indoles 4, including the standard deviation. A fungicidal effect is Pad Software, San Diego, CA, version 5.02 for Windows).
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Paper
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4. Conclusions
Since the 3-selenocyanate-indoles 4a and 4b are non-irritant to
the mucous membrane, these compounds could be employed
for the development of formulations to treat chronic wound
infections caused by Candida spp. and dermatophytes of the
genera Trichophyton and Microsporum. In the same way, they
could be used for the treatment of co-infections caused by
Candida and S. aureus, where higher concentrations of compounds
could be used to tackle infections without concerns regarding
genotoxicity. Around 2% of the population in developing countries
will develop chronic skin wounds, and the costs of treating these
infections is close to US$ 25 billion per year just in the United
States.15 Therefore, formulations containing 3-selenocyanate-
indoles might be further applied to preclinical studies in models of
superficial and mucocutaneous infections.
´
F. Piscitelli, E. Hamel, G. De Martino, R. Matesanz, J. F. Dıaz,
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Conflicts of interest
The authors declare no conflict of interest.
´
´
15 V. W. Soo, B. W. Kwan, H. Quezada, I. Castillo-Juarez, B. Perez-
´
´
´
Eretza, S. J. Garcıa-Contreras, M. Martınez-Vazquez, T. K. Wood
Acknowledgements
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and R. Garcıa-Contreras, Curr. Top. Med. Chem., 2017, 17,
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This work was supported by Brazilian agencies: the study was
financed in part by the Coordenaçao de Aperfeiçoamento de
16 G. P. Silveira, E. E. Silva, P. A. Pereira, N. Londero,
J. B. Azeredo and A. L. Braga, presented in part at 39th
Annual Meeting of the Brazilian Chemical Society, Goiania
Convention Center/Brazil, June, 2016.
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18 L. Sancineto, C. Tidei, L. Bagnoli, F. Marini, E. J. Lenardao
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´
Pessoal de Nıvel Superior (CAPES) and Conselho Nacional de
ˆ
´
´
`
Desenvolvimento Cientıfico e Tecnologico (CNPq) (Finance
˜
Code 001), and Fundaçao de Amparo a Pesquisa do Estado do
Rio Grande do Sul (FAPERGS – EDITAL 04/2016 – PRONUPEQ
2016). A. M. Fuentefria and M. H. Vainstein are grateful to
CNPq for their research fellowships.
˜
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