MEDICINAL CHEMISTRY RESEARCH

Med Chem Res DOI 10.1007/s00044-014-1258-8

ORIGINAL RESEARCH

Comparison of anti-Candida albicans activities of halogenomethylsulfonyl derivatives Małgorzata Bondaryk • Zbigniew Ochal Monika Staniszewska

•

Received: 6 June 2014 / Accepted: 11 September 2014 Ó Springer Science+Business Media New York 2014

Abstract A rapidly growing resistance of Candida spp. requires a search for bioactive compounds with fungicidal or fungistatic activity. In this context a characteristics and comparison of antifungal properties of 19 sulfone derivatives were conducted. MICs of the Compounds were determined using the M27-A3 protocol following CLSI recommendations. The SAP expression was analyzed using RT-PCR; relative quantification was normalized against ACT1 in cells grown in YEPD and on Caco-2. 79 % of sulfone derivatives (15 out of 19) exhibited an activity against Candida albicans in the tested concentrations. While the addition of both chlorine and bromine atoms to halogenomethylsulfonyl groups stimulates sulfone’s antifungal activity, a chlorine atom more effectively up-regulates antifungal properties of the tested sulfones. The insertion of a fluorine atom has a binary effect on antifungal properties of the tested sulfones. The fluorine atom enhances anti-Candida properties when introduced to the aromatic ring, while its presence in halogenomethylsulfonyl group generally lowers the Compound’s efficiency. The deletion of particular SAP genes resulted in an increased susceptibility of C. albicans toward sulfones indicating the role of this gene family in resistance mechanisms. Furthermore, RT-PCR analysis demonstrated that sulfone derivatives inhibit the SAP2 expression but not that of SAP7. M. Bondaryk  M. Staniszewska (&) Independent Laboratory of Streptomyces and Fungi Imperfecti, National Institute of Public Health-National Institute of Hygiene, Chocimska 24, 00-791 Warsaw, Poland e-mail: [email protected] Z. Ochal Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

Keywords Candida albicans  Resistance  SAP  Sulfone derivatives Abbreviations CLSI Clinical Laboratory Standards Institute DMSO Dimethyl sulfoxide EMEM Eagle’s minimum essential medium FCS Fetal calf serum MIC Minimal inhibitory concentration PBS Phosphate-based saline RPMI 1640 Roswell Park Memorial Institute Medium RT-PCR Reverse transcription polymerase chain reaction SAP Secreted aspartic proteases YEPD Yeast extract-peptone-dextrose growth medium XTT Sodium 30 -[1-(phenylaminocarbonyl)- 3, 4-tetrazolium]-bis (4-methyloxy-6-nitro) benzene sulfonic acid hydrate

Introduction In healthy individuals Candida albicans exists as a component of the normal microflora inhabiting mucosal surfaces of the urogenital, gastrointestinal, and vaginal tracts, as well as in the oral cavity without giving disease symptoms. However, in immunocompromised hosts this opportunistic pathogen causes superficial and systemic infections (Williams et al., 2013). Infections caused by Candida spp. are associated with a high morbidity and mortality (30–80 % of cases) and therefore remain a major problem worldwide. Thus, the pathogenic adaptation of C.

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albicans has been the topic of extensive investigations (Dos Santos, 2010; Eggimann et al., 2011). Like most pathogens, C. albicans possess a set of attributes that help the fungal cells to escape the host immune responses and cause a successful infection. These virulence factors include rapid switching from one phenotypic form to another, ability to undergo a reversible morphogenetic transition, adhesion to host cells, biofilm formation, and production of hydrolytic enzymes (Dos Santos, 2010; Kim and Sudbery, 2011; Nobile et al., 2012; Schild et al., 2011). Among the hydro-lytic enzymes, secreted aspartic proteases (Saps) are considered to be key virulence determinants of C. albicans, since these enzymes contribute to a wide range of fungal physiological processes, fungal-host interactions, as well as adhesive and invasion capabilities (Dos Santos, 2010). Saps are thus considered as new potential drug targets. The exposure of Candida spp. to antifungal agents is regarded as an environmental stress affecting the expression of the genes encoding virulence factors (i.e., adhesisns, Saps). Therefore, the immediate response of Candida spp. to antimicotics may represent a drug tolerance which leads to the drug resistance (Cannon et al., 2007; Copping et al., 2005; Costa et al., 2010). The rapidly growing antifungal resistance of Candida spp. remains a major threat to public health worldwide (Pfaller, 2012). Despite the introduction of new antimicotics, the overall number of drug-resistant C. albicans strains has been increasing. Rates of the reduced antifungal susceptibility or resistance ranging from \5 to [30 % have been reported (Eggimann et al., 2011). Therefore, the design of more effective antimicrobial agents is an important aspect in the management of fungal infections. Sulfone derivatives represent a group of synthetic compounds with antifungal activity which may meet above challenges. The sulfone group constitutes an important core found in numerous biologically active compounds with a wide range of biological activity, including anti-tumor, and anti-inflammatory properties (Xu et al., 2011). Among these derivatives many aromatic compounds bearing halogenomethylsulfonyl groups exhibit herbicidal as well as fungicidal activities (Ochal and Kamin´ski, 2005). The present study was focused mainly on the investigation of a large group of new synthesized sulfone derivatives for their anti-Candida activity. Additionally, based on a susceptibility assay, one Compound (as the most effective from the tested ones) was evaluated for its impact on the expression of C. albicans key virulence determinants, namely SAP. To test whether sulfone had an effect on virulence, we used an in vitro model of epithelial candidiasis based on Caco-2 (ATCC HTB27, LGC, Poland) for infections with this fungus pretreated with sulfone.

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Materials and methods The sulfone derivatives evaluated in the present study are listed in Table 1. The tested Compounds were synthesized at the Faculty of Chemistry of the Warsaw University of Technology (Poland). The synthetic pathway for the synthesis of the target compounds had been previously described (Ochal and Kamin´ski, 2005; Patent PL, P.408200, 2014). Strains and culture conditions Strains used throughout the study are listed in Table 2 (Gillum et al., 1984; Lermann and Morschha¨user, 2008; Liu et al., 1994; Lo et al., 1997; Staib et al., 2008). Strains were stored on ceramic beads in a Microbank tube (Prolab Diagnostics, Canada) at -70 °C. Prior to the respective examinations, routine culturing of strains was conducted at 30 °C for 18 h in YEPD (Ness et al., 2010). Susceptibility testing Susceptibility of C. albicans to the tested Compounds was analyzed using the standard CLSI microdilution protocol M27-A3 following CLSI recommendations (CLSI, 2008; Staniszewska et al., 2014b). The inoculum of 1–8 9 103 yeast cells/ml density diluted in PBS was incubated in increasing concentrations of each Compound (from 0.0313 to 160 lg/ml) diluted in RPMI (Life Technologies, Inc). Initially, the tested Compounds were prepared with the stock solution of 1,600 lg/ml dissolved in water with dimethylsulfoxide (DMSO) at 9 % (v/v). Then, the fungal blastoconidial suspension and the antifungal agent (final dilution 1:100) were dispensed into 96-well microplates (Sarstedt, Germany). The plates were incubated for 48 h at 35 °C with agitation. The reading was performed after 18 and 48 h of incubation, respectively. MIC, namely the lowest Compound concentration for which the well was optically clear, was determined visually after 48 h of incubation at 35 °C. The DMSO concentration was maintained at 0.09 % (v/v) in all the experiments, including the control ones. At this concentration, DMSO was unable to inhibit the growth of C. albicans. In order to validate our experiments, the MIC value of 1 lg/ml was obtained for amphotericin B (Sigma-Aldrich, USA). The amphotericin B was diluted in DMSO (1,600 lg/ml) to be subsequently used in the assay as a reference antifungal at the concentration of 1 lg/ml (100 % inhibition). The tests were performed in triplicate and repeated in three independent assays.

SO2CH2Cl

SO2CH2Cl

SO2CH2Cl

SO2CH2Cl

2

3

4

5

H3C

N N

N

H N

O

N

H N

Cl

Aromatic ring

Moiety

SO2CH2Cl

Ar

Z

1

No.

N

H N

CF 3

SH

N

C16H15ClN2O3S

C9H6ClN2O2SF3

C8H7ClN2O2S2

C7H6ClN3O2S

C7H6Cl2O2S

Chemical formula

Table 1 The sulfone derivatives evaluated in this study

350.82

298.67

262.73

231.66

225.09

(g/mol)

Mol mass

170–171

200–201

265–266

154–155

109–111

( C)

o

Melting temp.

76

92

88

77

88

(%)

Yield

54.78

36.19

36.57

51.85

37.35

4.31

2.02

2.69

7.01

2.69

7.88

9.38

13.49

13.44

54.73

36.15

36.52

51.81

37.22

4,42

1,98

2,62

6,98

2,56

%H

%C

%N

%C

%H

Assayed values

Calculated values

Elemental analysis

7.83

9.32

13.44

13.39

%N

20

20

[160

20

20

SC5314

MIC (lg/ml) after 48 h

Med Chem Res

123

123

SO2CHCl2

SO2CCl2Br

SO2CCl2Br

7

8

9

N

NO2

NO 2

O

NH

NO2

Cl

Cl

NH

Aromatic ring

Moiety

SO2CH2Cl

Ar

Z

6

No.

Table 1 continued

Cl

Cl

C14H11BrCl2N2O4S

C7H3Cl3BrN2O4S

C7H4Cl3NO4S

C15H11Cl3N2O3S

Chemical formula

454.12

383.43

304.53

405.68

(g/mol)

Mol mass

163–165

164–165

90–91

165–167

( C)

o

Melting temp.

87

94

93

78

(%)

Yield

37.03

21.93

27.61

44.41

2.44

0.79

1.32

3.64

6.17

3.65

4.6

2.73

36.98

21.88

27.73

44.37

2,5

0,75

1,24

3,58

%H

%C

%C

%H

Assayed values

Calculated values %N

Elemental analysis

6.12

3.61

4.55

2.67

%N

[160

2a

1a

20

SC5314

MIC (lg/ml) after 48 h

Med Chem Res

SO2CCl2Br

SO2CBr2Cl

SO2CBr2Cl

11

12

13

NO2

NHNH2 NO2

NH2

NHNH2 NO2

CH3 N

NO2

Aromatic ring

Moiety

SO2CCl2Br

Ar

Z

10

No.

Table 1 continued

C7H6Br2ClN3O4S

C7H5Br2ClN2O4S

C7H6BrCl2N3O4S

C14H11BrCl2N2O4S

Chemical formula

423.46

408.45

379.02

454.12

(g/mol)

Mol mass

127–130

120–122

199–201

144–145

( C)

o

Melting temp.

92

82

96

88

(%)

Yield

19.85

20.58

22.18

54.04

1.43

1.23

1.6

4.33

9.92

6.86

11.09

11.46

59.26

20.56

22.13

54.11

5.13

1,19

1,54

4,29

%H

%C

%N

%C

%H

Assayed values

Calculated values

Elemental analysis

11.63

6.83

11.04

11.42

%N

[16a

40

4

[160

SC5314

MIC (lg/ml) after 48 h

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123

123

SO2CHF2

SO2CHF2

SO2CF2Cl

SO2CHFCL

15

16

17

18

O 2N

O 2N

N

H N

Cl

N

CHF 2

CF 3

NO2

NO2

H N

Cl

NH2

Aromatic ring

Moiety

SO2CHF2

Ar

Z

14

No.

Table 1 continued

C7H4Cl2FNO4S

C9H5ClF4N2O2S

C9H5F5N2O2S

C7H3ClF2N2O6S

C7H6F2N2O4S

Chemical formula

288.08

316.66

300.2

316.63

252.19

(g/mol)

Mol mass

67–68

123–125

161–162

132–134

170–172

( C)

o

Melting temp.

92

86

74

70

87

(%)

Yield

29.18

34.14

36.01

26.55

33.34

1.4

1.59

1.68

0.96

2.4

4.86

8.85

9.33

8.85

11.11

29.22

34.11

36.04

26.61

33.29

1.46

1.62

1.63

0.92

2.46

%H

%C

%C

%H

Assayed values

Calculated values %N

Elemental analysis

4.84

8.78

9.31

8.91

11.08

%N

20

80

[160

160

20

SC5314

MIC (lg/ml) after 48 h

Med Chem Res

40 16.68

According to the supplier’s guidelines, monolayers of the colon-adenocarcinoma-derived cell line were maintained in a humidified incubator at 37 °C in 5 % CO2. For the sake of the experiment, 1.2 9 105 of Caco-2 cells/ml were seeded into 24-well-plates (Corning, USA) and cultured in the EMEM medium (10 % FCS, 1 mM pyruvic acid, without antibiotics or antifungal agents) up to 18 h. Next, after 18-h post seeding the Caco-2 monolayers were inoculated with 105 log phase yeast cells of the C. albicans. After 18 h of incubation Caco-2 was lysed by adding sterile water, in the result of which the C. albicans cells were recovered (Staniszewska et al., 2014b). Pre-treatment of C. albicans cells with dichloromethyl4-chloro-3-nitrophenylsulfone—compound no. 7

33.42 1.61

16.73

1.58 79.7

33.47

Yeast cells of C. albicans from an overnight culture grown on YEPD at 30 °C were suspended in PBS (of final density 105 cells/ml) and pre-incubated with the tested Compound at the concentrations of 0.5, 0.25, and 0.125 lg/ml for 2 h. Afterwards the cells were washed with PBS and used for tissue infection assay.

146–147

RT-PCR analysis to assess the affect of dichloromethyl4-chloro-3-nitrophenylsulfone—compound no. 7 on the genes expression: SAP2 and SAP7

Adapted from reference (Staniszewska et al., submitted)

N

N

C7H4F3N3O2S H N

Aromatic ring Moiety

SO2CF3 19

Ar Z No.

Table 1 continued

a

SC5314 %C %H

%N

%H

Cultivation and infection of Caco-2 cell line (ATCC HTB27, LGC, Poland)

251.18

(g/mol)

%C

Calculated values (%) ( C)

Elemental analysis

o

Chemical formula

Mol mass

Melting temp.

Yield

Assayed values

%N

MIC (lg/ml) after 48 h

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The expression assay was performed as we previously described (Staniszewska et al., 2014b). Briefly, in the case of the untreated blastoconidia, RNA was isolated from the cells after 18 h growth in YEPD at 30 °C. Additionally, the cells after having been grown in the YEPD medium were washed with water and following which 200 ll of the suspension were added to 1,800 ll of the RPMI medium (the final density of 104 cfu/ml) and inoculated onto the Caco-2 monolayer. Incubation was conducted for 18 h at 37 °C until the RNA extraction. For the cells pre-treated with tested Compound, blastoconidia grown in YEPD (as above), having been washed with water, were suspended in the YEPD medium containing 0.5, 0.25, or 0.125 lg/ml of Compound. Then, after incubation with tested Compound, the cells were washed with water and resuspended in 2,000 ll of RPMI, in order to be afterwards inoculated onto the Caco-2 monolayer for 6, 18, and 48 h at 37 °C. The total RNA was isolated as previously described (Amberg et al., 2005) and stored at -70 °C. The firststrand cDNA synthesis was performed using the Enhanced Avian HS RT-PCR kit (Sigma-Aldrich, USA) according to the manufacturer’s instructions. 1 ll of the total RNA, and 1 ll of oligo (dT)23 (3.5 lM), and 1 ll of dNTP mix

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Med Chem Res Table 2 Candida albicans strains used in this study Strain

Parental strain

Relevant characteristicsa or genotype

Reference

SC5314

None

Prototrophic wild-type strain (wt)

SAP1MS4B

SC5314

sap1-1D::FRT/sap1-2D::FRT

(Gillum et al., 1984) (Lermann and Morschha¨user, 2008)

SAP2MS4B

SC5314

sap2-1::FRT/sap2-2::FRT

(Staib et al., 2008)

SAP12MS4B

SC5314

sap1D : : FRT/sap1D : : FRT; sap2-1D : : FRT/sap2-2D : : FRT

(Lermann and Morschha¨user, 2008)

SAP13MS4B

SC5314

sap1-1D : : FRT/sap1-2D : : FRT; sap3D : : FRT/sap3D : : FRT

SAP23MS4C

SC5314

sap2-1D : : FRT/sap2-2D : : FRT; sap3D : : FRT/sap3D : : FRT

SAP3MS4B

SC5314

sap3D::FRT/sap3D::FRT

SAP123MS4C

SC5314

sap1D : : FRT/sap1D : : FRT; sap2-1D : : FRT/sap2-2D : : FRT; sap3D : : FRT/sap3D : : FRT

SAP4MS4B

SC5314

sap4-1D::FRT/sap4-2D::FRT

SAP5MS4B

SC5314

sap5-1D : : FRT/sap5-2D : : FRT

SAP6MS4B

SC5314

sap6-1D : : FRT/sap6-2D : : FRT

Can16

CAI4

cph1::hisG/cph1::hisG-URA3-hisG

(Liu et al., 1994)

YLO323

CAI4

cph1::hisG/cph1::hisG(CPH1)

(Lo et al., 1997)

HLC52

CAI4

efg1::hisG/efg1::hisG-URA3-hisG

HLC54

CAI4

cph1::hisG/cph1::hisG efg1::hisG/efg1::hisG-URA3-hisG

HLC74

CAI4

efg1::hisG/efg1::hisG (EFG1)

HLC84

CAI4

cph1::hisG/cph1::hisG efg1::hisG/efg1::hisG (EFG1)

a

Apart from indicated features all strains are identical to their parental strain

(500 lM each dNTP) were added to each tube to obtain 10 ll volume. Priming at 50 °C was carried out for 10 min. Subsequently, 1 ll of Enhanced Avian AMV-RT (1U/ll) and 1 ll of 109 buffer for AMV-RT were added to each tube to obtain 20 ll volume. The RT reaction was carried out at 50 °C for 50 min. SAP2 (F-50 TCCTGATGTTAAT GTTGATTGTCAAG30 and R-50 TGGATCATATGTCCC CTTTTGTT30 ) and ACT1 (F-50 GACAATTTCTCTTT CAGCACTAGTAGTGA30 and R-50 GCTGGTAGAGACT TGACCAACCA30 ) sequences in the C. albicans genome as previously described (Naglik et al., 2008). Additionally primer set was designed based on the unique SAP7 sequence as follows: F-50 ATGGACACAGTGTGAAATA TGAAGTG30 and R-50 TCAGTGGAGGATGGACCATT AGA30 . ACT1 was used as a housekeeping control gene for normalization. The FastStart Essential DNA Green Master (Roche, Germany) was used for detection according to the manufacturer’s instructions. The RT-PCR was performed as previously described (Naglik et al., 2008) using Light Cycler 96 (Roche, Germany). After the initial denaturation at 95 °C for 15 min, for the next 45 cycles thermal cycling conditions during the experiments were subsequently: 94 °C for 15 s, followed by 1 min at 60 °C. In each run negative (water) and positive (DNA of C. albicans SC5314) controls were included. The CT values were provided from RT-PCR instrumentation and were imported into a Microsoft Excel 2010 spreadsheet. The relative quantification was calculated using the 2-DDCT method (Livak and Schmittgen, 2001), where DCT = Avg. SAP

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CT - Avg. ACT1 CT; and DDCT = DCTsample–DCTcalibrator. Finally, 2-DDCT was calculated. P values of B0.05 were considered statistically significant. The tests were performed in triplicate and repeated in three independent assays. The Wilcoxon signed-rank matched-pair test was performed to evaluate the statistically significant differences in the SAP expression in the cells grown in YEPD or during the Caco-2 invasion; as well as the differences between the Compound-treated strains and the drug free controls. P values of B0.05 were considered statistically significant.

Results In the study, the series of new Compounds bearing halogenomethylsulfonyl moieties based on benzene ring were synthesized according to the described procedure (Ochal and Kamin´ski, 2005; Patent PL, P.408200, 2014). The physical characteristics and elemental analyses data are presented in Table 1. The minimum inhibitory concentration (MIC) is the lowest concentration of an antimicrobial compound that inhibits the visible growth of a microorganism as indicated by the absence of turbidity (optically clear wells). MIC values of the Compounds against C. albicans strains were determined on the basis of microdilution method following CLSI recommendations. Initially, all Compounds were tested for their antifungal activity against C. albicans reference strain SC5314

Med Chem Res

(ATCC) (Table 1). Four Compounds had no activity against C. albicans wt in the tested concentrations (MIC [160 lg/ml). The remaining sulfone derivatives displayed moderate or good activity against C. albicans. Sulfones bearing the benzene ring were shown to inhibit C. albicans growth. Compound no. (1) possessed a good antifungal activity (MIC = 20 lg/ml). A further addition of a chlorine atom, respectively, in the sulfone and nitro moieties at the ortho position in the aromatic ring significantly increased sulfone’s antifungal activity, lowering MIC values from 20 lg/ml (Compound no. (1)) to 1 lg/ml (Compound (7), (Staniszewska et al., submitted)). However, a further addition of a bromine atom to the sulfone moiety in Compound no. (8) (Staniszewska et al., submitted) lowered the sulfone’s efficiency (2-fold) toward the fungal cells compared with Compound no. (7), suggesting superior role of a chlorine atom in antifungal activity. On the other hand, the antifungal activity comparison of Compounds: no. (7) and no. (18) showed that the nitro moiety at the ortho position in the aromatic ring had no effect to this process. However, substitution of one chlorine atom with a fluorine (Compound no. (18) in the halogenomethylsulfonyl moiety, decreased activity of the Compound (20-fold) compared with Compound no. (7). In the case of Compound no. (15), further replacement of chlorine with fluorine atoms strongly diminished Compounds’ activity against C. albicans (MIC [160 lg/ml). The comparison of the activity of Compounds: no. (8) and no. (11) pointed out those substitutions of chlorine in the aromatic ring influence the antifungal activity. Thus, the replacement of the chlorine with hydrazine moiety in the benzene ring gave 2-fold lowered anti-Candida activity of Compound no. (11) compared with Compound no. (8). Whereas, substitution of a chlorine with bromine atom in the halogenomethylsulfonyl moiety as seen in Compound no. (13) lowered its activity 4-fold compared with Compound no. (11). Based on the activity comparison we found that sulfones bearing the benzotriazole ring (Compound no. (2)) were as effective against C. albicans wt as sulfones containing the benzene ring with the same halogenomethylsulfonyl moiety (Compound no. (1)). Furthermore, the substitution of one chlorine (Compound no. (2) with fluorine (Compound no. (19)) in the halogenomethylsulfonyl moiety resulted in decreased (2-fold) antifungal activity. The activity comparison of benzimidazole containing sulfonyl groups demonstrated that presence of the sulfhydryl moiety in the imidazole ring fully diminished antiCandida properties of Compound no. (3) (MIC [160 lg/ ml), despite the presence of a chlorine in the halogenomethylsulfonyl group. While, substitution of the sulfhydryl moiety with a trifluoromethyl group in the benzoimidazole ring (Compound no. (4)) or with the

phenoxymethyl moiety as in the case of Compounds: no. (5) and no. (6), strongly increased sulfones’ antifungal activity (MIC = 20 lg/ml). On the contrary, introduction of a fluorine to the halogenomethylsulfonyl moiety of Compound no. (17) resulted in 4-fold lowered activity compared with Compound no. (4). Whereas, substitution of a chlorine with fluorine atom in the halogenomethylsulfonyl moiety of Compound no. (16) fully diminished antiCandida activity. Overall, the nitroaniline-based sulfones exhibited a good activity against C. albicans wt. On the contrary, in the benzene-based sulfones, the substitution of chlorine and bromine with fluorine in the halogenomethylsulfonyl moiety in Compound no. (14) resulted in 2-fold higher antifungal activity compared with Compound no. (12). Furthermore, the additional benzene moiety in the aromatic ring fully diminished the antifungal activity nitroanilinebased sulfones as seen in Compounds: no. (9) and no. (10). Depending on the MIC values against the reference strain, the activity of Compounds was further tested against the attenuated strains (Dsap and Defg1/cph1). Among all presented sulfones, bromodichloromethyl-4-chloro-3-nitrophenylsulfone (Compound no. (8)), dichloromethyl-4chloro-3-nitrophenylsulfone (Compound no. (7)), and chlorodibromomethyl-4-hydrazino-3-nitrophenylsulfone (Compound no. (13)) were evaluated against the attenuated strains in our previous work (Staniszewska et al., submitted). In this study, one of the tested Compounds named 4-(dibromochloromethylsulfonyl)-2-nitroaniline (Compound no. (12)) with good anti-Candida activity against wt, was chosen for further antifungal activity assay with respect to the attenuated C. albicans strains. As indicated in Table 3, a single deletion of the particular SAP gene increased C. albicans susceptibility (2.5–5-fold) toward Compound no. (12) compared with wt. Although the deletion of two SAP genes led to 10–20-fold increased susceptibility compared with wt, a further susceptibility enhancement was not observed in the triple SAP mutant. Furthermore, the susceptibility of the strains attenuated in the key morphogenesis regulators (Efg1 and Cph1) was evaluated. Deletion of EFG1 or both EFG1 and CPH1 led to increased susceptibility of the mutants toward Compound (12). While reintroduction of the functional EFG1 copy did not contribute to resistance of the strain. Interestingly, a deletion of CPH1 led to reduced susceptibility of the mutant toward tested agent. Whereas reintroduction of one functional copy of CPH1 resulted in the increased susceptibility compared with wt (2.5-fold) and the Dcph1 mutant (5-fold). Impact of sulfone derivatives on the SAP expression Compound no. (7) containing dichloromethylsulfonyl moiety, at the para position of the benzene ring with the

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Med Chem Res Table 3 Activity of 4-(dibromochloromethylsulfonyl)-2-nitroaniline (Compound (12)) against the C. albicans wt and attenuated strains (the Dsap and morphogenesis mutants) Strain

MIC[lg/ml]

Susceptibility changesa,

SC5314

40

Control

SAP4MS4B

8

5-fold :

SAP5MS4B SAP6MS4B

8 8

5-fold : 5-fold :

SAP1MS4B

16

SAP2MS4B

8

5-fold :

SAP3MS4B

8

5-fold :

SAP12MS4B

2

20-fold :

SAP13MS4B

4

10-fold :

SAP123MS4C

20

2-fold :

SAP23MS4C

2

20-fold :

Can16

80

2-fold ;

YLO323

16

2.5-fold :

HLC52

20

2-fold :

HLC54

20

2-fold :

HLC74

16

2.5-fold :

HLC84

16

2.5-fold :

b

2.5-fold :

a

: Stands for fold increase in susceptibility to (12) compared with the control strain b ; Stands for fold decrease in susceptibility to (12) compared with the control strain

chlorine and nitro, substituents attached to C-1and C-2 positions respectively displayed remarkable activity against C. albicans, fully inhibiting planktonic growth wt SC5314 at concentration of 1 lg/ml, as indicated by the absence of turbidity (optically clear wells, Table 1, (Staniszewska et al., submitted)). Dichloromethyl-4chloro-3-nitrophenylsulfone (Compound no. (7)) was proven to be the most active against C. albicans wt among the tested sulfones and therefore chosen for the gene expression study. As estimated in our antifungal assay (Table 3, (Staniszewska et al., submitted)), the strains attenuated in the SAP genes were found to be more sensitive compared with wt. Furthermore, disturbance of the genes from SAP46 family increased the strain susceptibility by 5-fold of that of wt. Therefore, the RT-PCR system was applied in order to investigate the impact of Compound no. (7) to the expression of SAP2 (Table 4) and SAP7 (Table 5) in the triple Dsap456 mutant during Caco-2 invasion after transient exposure to Compound (2 h) in series of wt subinhibitory concentrations: 0.5, 0.25, and 0.125 lg/ml. Following this limited exposure, Compound no. (7) was removed according to previously described procedure (Ellepola et al., 2014). The Candida cells were washed, resuspended in 2,000 ll of RPMI and used for tissue infection. The effect of sulfone derivatives on cell viability was determined following sulfone removal. The tested

Table 4 Influence of dichloromethyl-4-chloro-3-nitrophenylsulfone (Compound (7)) on the SAP2 expression in C. albicans during the growth on Caco-2. The expression level values were normalized according to the 2-DDCT method Mean of the SAP2 mRNA transcript levelsa,

b

Strain

Medium/Time (h)

Tested concentration (lg/ml)

2-DDCT

SDc

Rd,

SC5314

YEPD/18

None

1.00Cal

±0.05

Control

Cal

Caco-2/18 Dsap456

YEPD/18

None

Caco-2/18 Caco-2/6

±0.36

Control

±3.21

Control

1.83

±0.49

ControlCal

0.70

±0.22

61.57 %

0.73

±0.23

60.12 %

Caco-2/48

0.60

±0.19

67.48 % 79.40 %

0.38

±0.10

Caco-2/18

0.35

±0.22

80.73 %

Caco-2/48

0.23

±0.17

87.53 %

1.67 1.29

±0.16 ±0.37

8.63 % 29.38 %

0.43

±0.07

76.43 %

Caco-2/6 Caco-2/18 Caco-2/48 a

1.00 4.84

Caco-2/18 Caco-2/6

0.5

e

0.25

0.125

Mean of the SAP2 mRNA transcript levels based on up to five biological replicates (at least three). Normalized to ACT1

b

The P value B 0.05: the Dsap456 strain was calibrated to SC5314 (the study calibrator); For antifungal activity assay: the compound-treated strains were compared to the non-treated control (Wilcoxon signed-rank matched-pair test)

c

±Standard deviation (SD)

d

R stands for the SAP2 expression reduction %

e

Cal stands for study calibrator

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Med Chem Res Table 5 Influence of dichloromethyl-4-chloro-3-nitrophenylsulfone (Compound 7) on the SAP7 expression in C. albicans during the growth on Caco-2. Expression level values were normalized according to the 2-DDCT method Mean of the SAP7 mRNA transcript levelsa,

b

Strain

Medium/Time (h)

Compound concentration (lg/ml)

SC5314

YEPD/18

none

Caco-2/18

Dsap456

YEPD/18 Caco-2/18

none

Caco-2/6 h

0.5

2-DDCT

Ic,

d

0.41

0.75

1.00Cal

Control

-0.94

1.92

1.00Cal

-1.23 -0.20

2.35 1.15

3.11 0.70

ControlCal

-1.49

2.81

2.45

3.52-fold

Caco-2/18

-1.62

3.07

2.68

3.85-fold

Caco-2/48

-1.58

2.99

2.60

3.74-fold

-1.82

3.53

3.07

4.42-fold

-0.53

1.44

1.26

1.81-fold

-1.26

2.39

2.08

3.00-fold

Caco-2/18

-1.63

3.10

2.69

3.87-fold

Caco-2/48

-1.62

3.07

2.68

3.85-fold

Caco-2/6 h

0.25

Caco-2/18 Caco-2/6 h

a

2-DCT

Mean DCT

0.125

Mean of the SAP7 mRNA transcript levels based on up to five biological replicates (at least three). Normalized to ACT1

b

The P value C 0.05: the Dsap456 strain was calibrated to SC5314 (the study calibrator); For antifungal activity assay: the compound-treated strains were compared to the non-treated control (Wilcoxon signed-rank matched-pair test)

c

I stands for SAP7 fold expression induction

d

Cal stands for study calibrators

sulfones interfered with the expression of SAP2 and SAP7 without affecting cells’ viability after 2-h of incubation as was shown in our recent studies including different set of strains (Bondaryk et al., 2014). The impact of Compound no. (7) to SAP2 and SAP7 is presented in Table 4 and Table 5, respectively.

Discussion The present work demonstrated that sulfone derivatives can be used as possible lead compounds for the development of the potential antifungal agents. Most of them (15 out of 19) exhibited a satisfactory activity against C. albicans under the tested conditions. The performed biological studies shown that the activity varies between the Compounds bearing, respectively, benzene, nitroaniline, or benzoimidazole aromatic ring. Although a wide variety of benzimidazole derivatives have been previously described for their antifungal activity (Kawasaki et al., 2003; Narasimhan et al., 2010), our results showed that benzoimidazolebased halogenomethyl sulfones are the weaker agents against C. albicans wt SC5314 among the all Compounds tested. While benzene-based Compounds were the strongest inhibitors of fungal strains. However, our results indicated that various substitutions of halogenometyl moiety in both aromatic ring and sulfone group play a central role in the Compound’s activity. Previously,

derivatives of phenyl tribromomethyl sulfone were reported for their potential antifungal activity (Borys et al., 2012). Here we showed that the addition of both chlorine and bromine atoms in the halogenomethylsulfonyl groups stimulates the sulfones’ antifungal activity. However, out of these two atoms, the chlorine substituent more effectively up-regulated the Compounds’ antifungal properties. Moreover, substitution of the hydrazino moiety in the aromatic ring with a chlorine substituent also enhanced the sulfones’ antifungal activity which is in agreement with the study of (Korzyn´ski et al., 2014). Furthermore, according to the latter authors (Korzyn´ski et al., 2014); the sulfone derivatives bearing trifluoromethyl group were found to be superior to their 2-methyl-substituted analogues in the antifungal activity. Interestingly, we found that the insertion of fluorine atoms has a binary effect on the antifungal properties of the sulfones tested. Our results indicated that a fluorine atom enhances anti-Candida properties when introduced to the aromatic ring, while its presence in the halogenomethylsulfonyl group generally lowers Compound’s efficiency with exception to the nitroaniline-based sulfones. It is known that the blockage of C. albicans virulence factors should attenuate the infection process (Dos Santos, 2010; Naglik et al., 2008). Using the genetic alternations in the genes encoding Sap izoenzymes and the morphogenesis regulators (Efg1 and Cph1) we tested whether these genes could have an impact on C. albicans’ resistance to the

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tested sulfones. Our studies showed that the deletion of EFG1 but not CPH1 contributed to the increased susceptibility of C. albicans toward Compound 7(12) compared with wt. The latter indicated that the mechanism underlying this phenomenon may be dependent on the cAMP-dependent PKA pathway which is regulated by the EFG1 transcription factor (Watamoto et al., 2010). The Sap inhibitors have long been discussed as novel antimicotic agents for the treatment of candidiasis (Dos Santos, 2010; Puri et al., 2012). In the current study, we demonstrated that the SAP alterations increase susceptibility of C. albicans toward sulfones. However, effective environmental and stress adaptation represent an essential feature of all pathogens (Ene et al., 2012). Similarly, C. albicans can utilize alternative Sap proteins when one is compromised, removed or lost (Hube and Naglik, 2001). The Sap expression assays after exposure to antimicrobials are based on the in vitro Sap2 activity, the dominant secreted aspartic protease in vitro (Hube et al., 1994; Kathwate and Karuppayil, 2013; Wu et al., 2000). It has been demonstrated that virulence factors of C. albicans were significantly reduced after limited or transient exposure to subcidal concentrations of antifungal agents (Ellepola et al., 2014). The inhibition of SAP4-6 involved in C. albicans’ morphogenesis and adhesion process (Naglik et al., 2008; Sardi et al., 2013; Tsai et al., 2013) represents a promising strategy for the design of new agents with effective antifungal abilities targeting Candida spp. As previously described (Naglik et al., 2008; Dos Santos, 2010) SAP4-6 strongly impacts full-virulence of C. albicans phenotype in systemic infections. Using the genetic alternations in SAP4-6 we analyzed whether these aspartic proteases could have an impact on C. albicans’ resistance to the sulfone tested. In this study, treatment of the Dsap456 mutant with subinhibitory concentrations of Compound no. (7) demonstrated the differential transcriptional level of SAP2. During the study, C. albicans compensated the functional loss of SAP4-6 subfamily by up-regulating the alternative SAP2 expression in YEPD and during Caco-2 invasion. While, the SAP2 transcript was detected in C. albicans YEPD pre-cultured cells of both strains tested, SAP2 tended higher in Dsap456 compared with wt. Likewise, during Caco-2 invasion, higher levels of SAP2 were detected in Dsap456 compared with SC5314. Similarly, (Naglik et al., 2008) demonstrated that C. albicans can utilize alternative Sap proteins when one isoenzyme becomes compromised in RHE model. Next, we demonstrated that the transient exposure to Compound no. (7) nullified the compensative SAP2 over-expression in the C. albicans Dsap456 mutant during Caco-2 invasion, down-regulating the expression of this gene up to 87.53 %. Despite prolonged Caco-2 invasion time (from 6 to 48 h); the SAP2 expression was still

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significantly inhibited in C. albicans suggesting strong proteins-inhibiting post-antifungal effect (PAFE). Previously, PAFE impact on Candida spp. virulence factors was demonstrated for nystatin (Ellepola et al., 2014). The carryover effect of sulfone following its removal was minimal due to washing and centrifugation procedures which were shown to reduce agents concentration by 10,000-folds (Ellepola et al., 2014). Whereas, viable counts of the control and treated samples performed each time following sulfone removal showed no significant difference in cells growth. The latter eliminate possibility that the SAP2 inhibition was due to low post-exposition of the yeast cell survivability. To date, it was shown that the SAP7 expression responds to the challenge of the human blood environment (Staniszewska et al., 2014a; Taylor et al., 2005). We previously demonstrated (Staniszewska et al., 2014a) that compensatory up-regulation of SAP7 occurs in the Dsap mutant under serum influence. Moreover, according to (Aoki et al., 2012) pepstatin A did not have any effect on the Sap7 activity despite inhibiting other Saps suggesting a role of this protein in drug resistance. Therefore, in this study we additionally assayed the influence of Compound no. (7) on the SAP7 expression in the Dsap456 mutant. Interestingly, the SAP7 expression was induced by the tested sulfone in subinhibitory concentrations. Although data was not statistically significant (P [ 0.05), the induction of the SAP7 mRNA levels might represent a compensation mechanism for reduced activity of SAP2 caused by Compound no. (7). Sap7 is the most divergent and least known member of the whole SAP gene family (Dos Santos, 2010). (Aoki et al., 2012) demonstrated that amino acid residues in close proximity to the enzyme’s active site affect Sap7 insensitivity to pepstatin A. Among 9 amino acids identified, M242A and T467 prohibit pepstatin A to access to the active site of Sap7. The remaining 7 amino acid residues might therefore be involved in resistance mechanisms toward different compounds such as sulfone derivatives. Here, we discussed the effect of varying fungal sensitivity upon the transformations methods in C. albicans, including treatment with LiOAc and electroporation. To prove that the observed phenotypes of the mutants were caused by the lack of a functional genes of transcriptional factors, we used control mutant strains in which the EFG1 or CPH1 genes had been reintroduced into deficient strains. On the other hand, the differences in the locus site for URA3 reintegration may have somehow affected the ability of the latter mutants to control other genes expression. The morphogenesis mutants: Defg1, Dcph1, and Dcph1 Defg1 used in the current study had been derived from the wild type (Liu et al., 1994; Lo et al., 1997) using a technique that makes strains heterozygous for the URA3 gene (Negredo et al., 1997; Wilson et al., 1999). As reported (Forche

Med Chem Res

2014) heterozygosity itself is advantageous and maintains the high genetic diversity. However, the authors (Brand et al., 2004; Cheng et al., 2003; Garcı´a et al., 2001; Jackson et al., 2007; Xu et al., 2014) documented several shortcomings of this method. As noted briefly, URA3 expression levels can affect virulence and are susceptible to chromosome position effects, thereby complicating the analysis of strains constructed with URA3 as a selectable marker. However, recently it was demonstrated that the URA3 genes were apparently not necessary for C. albicans to adhere to and colonize the alimentary tract, form hyphae, infect oro-oesophageal and gastric tissues, and kill the mice (Balish, 2009). The SAT1 nourseothricin resistance marker cassette was used for gene disruption of SAP1-6 in the SC5314 background (Lermann and Morschha¨user, 2008; Staib et al., 2008). The SAT1 flipping method relies on the use of a cassette that contains a dominant nourseothricin resistance marker (caSAT1) for the selection of integrative transformants (Reuss et al., 2004). However, this cassette leaves a short FRT sequence in the genome after the excision of the marker (Xu et al., 2014). Nevertheless, the SAT1 flipping method provides a highly efficient method for gene disruption in the Candida spp. wild-type strains and remains to be used (Jandric et al., 2013). As reviewed (Biswas et al., 2007), the development of these moleculat tools greatly accelerated the elucidation of virulence and novel antifungal targets of C. albicans. In conclusion, a series of 19 sulfone derivatives have been screened against C. albcans with amphotericin B as a control drug. Generally, the presence of chlorine and bromine atoms in the halogenomethylsulfonyl groups highly promoted anti-Candida activity. The synthesized Compounds showed moderate to good antifungal activity, while four of them were highly active. Introducing of sulphonyl group observed for compounds resulted in chlorine atoms very closely located to carbon in halogenomethylsulfonyl groups, thereby prompting stability without affecting (such as halogenalkyl derivatives) either the amino, hydroxyl, or thiol groups (data not shown). The sulfone derivatives inhibited the growth of fungal strains but the compounds did not cause cytotoxicity against mammalian cell line. XTT analysis confirmed no cytotoxicity of sulfones (containing different halogen atoms among them chlorine atom) to proliferating Vero cells (ATCC CCL-81) at tested concentrations below 16 lg/ml (data not shown). Thus, sulfones affected the fungal protein synthesis mechanism and did not have any reported side-effects to the mammalian cells. The additional screening of the Compound no. 7’ influence on strains containing genetic alternations indicated the role of SAP genes, particularly SAP7 and morphogenesis regulators (EFG1, CPH1) in fungal resistance mechanisms.

Acknowledgments The work was supported by the research project of the National Science Centre, Project DEC-2011/03/D/NZ7/06198. We are extremely grateful to many colleagues and all the individuals who were generous with their advice, and provided us with strains and reagents; Professor Hsiu-Jung Lo from National Health Research Institute in Zhunan (Taiwan) with the following strains: Can16, YLO323, HLC52, HLC54, HLC74, and HLC84; Professor Joachim Morschha¨user from University of Wu¨rzburg (Germany) with following strains: SAP1MS4B, SAP2MS4B, SAP12MS4B, SAP13MS4B, SAP23MS4C, SAP3MS4B, SAP4MS4B, SAP5MS4B, SAP6MS4B, and SAP456MS4B. Conflict of interest of interest.

The authors declare that they have no conflict

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