Heterocycle thiazole compounds exhibit antifungal activity through increase in the production of

27 Pages·2017·1.03 MB·English

AAC Accepted Manuscript Posted Online 22 May 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.02700-16
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
1 Heterocycle thiazole compounds exhibit antifungal activity through increase in the
2 production of reactive oxygen species in Cryptococcus neoformans/Cryptococcus gattii
3 species complex
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5 Nívea Pereira de Sá1, Caroline Miranda de Lima1, Cleudiomar Inácio Lino2, Paulo Jorge
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6 Sanches Barbeira2, Ludmila de Matos Baltazar1, Daniel Assis Santos1, Renata Barbosa de o
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7 Oliveira3, Eleftherios Mylonakis4, Beth Burgwyn Fuchs4, Susana Johann1*. lo
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10 1 Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de t
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11 Minas Gerais, Belo Horizonte, MG, Brazil. /a
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12 2 Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas .a
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13 Gerais, Belo Horizonte, MG, Brazil. .o
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14 3 Departamento de Produtos Farmacêuticos, Faculdade de Farmácia da UFMG, /
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15 Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. A
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16 4 Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School, and il 1
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17 Brown University, Providence, RI, United States. 0
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19 *Corresponding author: u
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20 Susana Johann, Departamento de Microbiologia, Instituto de Ciências Biológicas,
21 Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, PO Box 486, 31270-901,
22 Belo Horizonte, MG, Brazil. Tel.: [phone]; Fax: [phone]; Email:
23 [email protected]
24
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25 ABSTRACT
26 Human cryptococcosis can occur as a primary or opportunistic infection and
27 develops as an acute, sub-acute, or chronic systemic infection, involving different organs of
28 the host. Given the limited therapeutic options and the occasional resistance to fluconazole,
29 there is a need to develop novel drugs for the treatment of cryptococcosis. In this report, we
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30 describe promising thiazoles 1, 2, 3, and 4 and explore their possible modes of action o
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31 against Cryptococcus. To this end, we show evidence of interference in the Cryptococcus lo
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32 antioxidant system. The tested compounds exhibited MIC ranging from 0.25 to 2 µg/mL d

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33 against C. neoformans H99 and KN99α strains. Interestingly, the knockout strains for cu- om

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34 oxidase and sarcosine oxidase were resistant to thiazoles. MIC values, mainly, of 1, 2, and t
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35 4 against these mutants were higher than for the parental strain. After treatment of C. /a
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36 neoformans ATCC 24067 (=C. deneoformans), and C. gattii L27/01 (=C. deuterogattii) .a
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37 with thiazoles we verified an increase of intracellular ROS. Also, we verified the .o
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38 synergistic interaction among thiazoles and menadione, which generates superoxides, with /
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39 FIC equal to 0.1874, 0.3024, 0.25, and 0.25 for the thiazoles 1, 2, 3, and 4, respectively. In A
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40 addition, thiazoles exhibited antagonistic interaction with FeTPPS. Thus, in this work we il 1
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41 showed that the action of these thiazoles is related to interference in the antioxidant system. 0
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42 These findings suggest that the oxidative stress may be primarily related to the b
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43 accumulation of superoxide radicals. u
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44 Keywords: antifungal, antioxidant, Cryptococcus, thiazoles, ROS, and RNS
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46
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47 INTRODUCTION
48 Cryptococcosis is an opportunistic or primary fungal infection caused by yeasts of
49 the genus Cryptococcus, mainly C. neoformans and C. gattii. These encapsulated fungi can
50 cause disease in many animals and humans, leading to severe clinical conditions, such as
51 meningoencephalitis (1). The treatment of the cryptococcosis caused by C. neoformans or
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52 C. gattii is very similar and includes the use of antifungal agents (azoles, polyenes, and o
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53 nucleoside analogues), cerebrospinal fluid drainage, and occasionally surgical resection (2, lo
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54 3). C. neoformans has a global distribution, while the various species within the C. gattii d

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55 species complex are geographically limited. The genotype AFLP4/VGI (=C. gattii s.s.) and om

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56 AFLP6/VGII (=C. deuterogattii) have a global occurrence, while AFLP5/VGIII (=C. t
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57 bacillisporus) is limited to the America's and genotype AFLP7/VGIV (=C. tetragattii) is /a
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58 limited to Sub-Saharan Africa and India. Clinical cases are also frequently reported from .a
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59 other continents, not only the America's and Africa (4, 5). Cryptococcosis is responsible for .o
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60 about 624,000 deaths per year worldwide (6), with universal geographic distribution, but /
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61 clinical cases are more common in the Americas and Africa (7, 8). A
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62 The antifungal options for the treatment against cryptococcosis is largely limited to il 1
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63 three agents: amphotericin B, 5-fluorocytosine (5-FC) and fluconazole (9). Infectious 0
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64 Diseases Society of America (IDSA) (2010) suggests specific recommendations for b
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65 cryptococcal meningoencephalitis treatment, can be managed successfully in the vast u
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66 majority of patients (10). However in underdeveloped countries like sub-Saharan Africa,
67 still rely on fluconazole monotherapy (11). In this case high doses of fluconazole are
68 necessary (10, 12). In addition, each of these drugs presents challenges. Amphotericin B is
69 noted for causing nephrotoxicity and thus is administered only as an initial therapeutic (13).
70 Renal failure was noted in 30% of the cases in a study of 707 patients receiving
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71 deoxycholate amphotericin B therapy (14), while liposomal formulations cannot be used in
72 most developing countries because of cost. Also, 5-FC resistance is considered common
73 and prevents its use as a monotherapy against cryptococosis (15). Fluconazole is used for
74 secondary prophylaxis, but cases of acquired resistance by C. neoformans have been
75 reported, resulting in treatment failures (16, 17, 18, 19). Overall, with the current
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76 therapeutic options, the mortality rate for the disease remains unacceptably high, as much o
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77 as 30% (15, 20). lo
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78 The thiazoles compounds, which possess sulfur and nitrogen, are widely distributed d

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79 in natural products and have promising biological activity, being a class of substances that om

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80 attracts great synthetic interest (21). Some studies have reported the synthesis of new t
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81 thiazoles with anti-inflammatory, antibacterial, anti-cancer, and anti-HIV activities (22, 23, /a
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82 24, 25). One of the groups involved in this work, investigated novel thiazoles heterocyclics .a
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83 compounds, which are easy to synthesize, have antifungal activity against C. gattii and C. .o
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84 neoformans with minimal inhibitory concentrations that range from 0.25 to 1.25 μg/mL and o/
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85 display low cytotoxicity in VERO cells and murine macrophages presenting IC50 up to 32
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86 μg/mL (26). In this previous study (26) we provided evidence that the thiazole fungicides 1, il 1
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87 2, 3, and 4 are promising compounds for the development of new drugs for the treatment of 1
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88 cryptococcosis. Building on our previous findings, in the present work we used a library of y

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89 mutants of C. neoformans H99 that aided in the investigation of mechanisms of fungal e
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90 inhibition of these new compounds. These experiments with mutants directed studies the
91 studies with oxidative stress.
92
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93 MATERIALS AND METHODS
94 Antifungal compounds. Thiazoles compounds 1, 2, 3, and 4 were synthesized according to
95 methodology previously described by Sá et al. (26) (Figure 1).
96 Strains and culture conditions. C. neoformans ATCC 24067 (AFLP2/VNIV, =C.
97 deneoformans according name proposed by Hagen et al.) (5), was obtained from the culture
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98 collection of the University of Georgia (Atlanta, GA, USA). C. neoformans KN99α and C. o
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99 neoformans H99 (AFLP1/VNI, both serotype A) were obtained from the culture collection lo
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100 of the Division of Infectious Diseases, Brown University (Providence, RI, USA). C. gattii d

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101 L27/01 (VNII, by PCR-fingerprinting using (GTG)5 primer, unpublished data) (= C. om

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102 deuterogattii according name proposed by Hagen et al. (5)) deposited in the Culture t
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103 Collection of Microorganisms and Cells of the Universidade Federal of Minas Gerais under /a
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104 code UFMG-CM-Y6141 (Belo Horizonte, MG, Brazil). All the yeast strains were stored .a
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105 frozen at -80ºC. In present work we will refer to the isolates of different molecular species .o
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106 as C. neoformans/C. gattii species complex, as a proposed by Kwon-Chung et al. (27). /
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107 In vitro susceptibility testing. Broth microdilution testing was performed in accordance A
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108 with the guidelines in the CLSI document M27-A3 (28). Susceptibility was determined by il 1
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109 the microbroth dilution method, which was performed in sterile flat-bottom 96-well 0
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110 microplates. Briefly, Cryptococcus neoformans species complex (H99, and KN99α) and C. b
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111 gattii (L27/01 =C. deuterogattii) were cultured in Yeast Peptone Dextrose (YPD) broth at u
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112 37°C for 48 h and the inoculum concentration was 2.5 × 105 cfu/mL. The thiazoles t
113 compounds 1, 2, 3, and 4 were dissolved in dimethyl sulfoxide (DMSO) (Sigma Aldrich,
114 St. Louis, MO, USA) and diluted in RPMI 1640 medium supplemented with L-glutamine
115 and buffered to pH 7.0 with 0.165 M morpholine propanesulfonic acid (MOPS) (Sigma
116 Aldrich, St. Louis, MO, USA). Compounds were tested at concentrations from 0.125 to 64
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117 µg/mL. Growth and sterility controls were also performed. Fluconazole (Sigma Aldrich, St.
118 Louis, MO, USA). and amphotericin B (Sigma Aldrich, St. Louis, MO, USA). were
119 included as the positive antifungal controls, and were tested from 0.125 to 64 µg/mL. The
120 plates were incubated at 35°C for 72 h. MIC values were determined using a
121 spectrophotometer (Bio-Rad Benchmark Plus spectrometer) at 595 nm.
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122 Screening of cryptococcal mutants. We screened the Madhani collection of C. o
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123 neoformans mutants available from the Fungal Genetic Stock Center (29) in search of lo
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124 strains with resistance to our group of compounds. Briefly, the library of 1,265 strains of C. d

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125 neoformans H99 mutants and H99 wild type were cultured in YPD broth at 30°C overnight. om

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126 The thiazoles compounds 1, 2, 3, 4 were dissolved in DMSO and diluted in RPMI 1640 and t
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127 evaluated at MIC (2, 0.25, 2, and 2 µg/mL, respectively) and 6x MIC concentrations by /a
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128 adding 200 µL to sterile flat-bottom 96-well microplates. The inoculum was added using .a
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129 stamp replicator. The plates were incubated at 37°C for 48 h. In all experiments were .o
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130 carried out controls with H99 and mutant isolates, and the isolates that did not present /
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131 similar growths to H99 at 30ºC and 37ºC were disregarded. Growth within the wells was A
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132 evaluated using a spectrophotometer (Bio-Rad Benchmark Plus spectrometer) at 595 nm. il 1
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133 Isolates that exhibited growth in the test compounds were further evaluated by broth 0
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134 microdilution testing to determine the MIC of the compounds against the mutant strain, b
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135 performed in accordance with the guidelines in the CLSI document M27-A3 (28). u
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136 Cellular ROS and RNS. The C. neoformans strain ATCC 24067 (=C. deneoformans) ,and
137 C. gattii strain L27/01 (=C. deuterogattii) were cultured in Sabouraud-dextrose agar during
138 48 hours at 37ºC. Microplate wells received 100 μL inoculum containing 1x105 cfu/mL in
139 RPMI medium without phenol red, then the probes were added. Briefly, the probe for
140 reactive oxygen species (ROS) (2',7'-dichlorofluorescein diacetate, Sigma Aldrich, St.
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141 Louis, MO, USA) and reactive nitrogen species (RNS) (dihidrorhodamine 123, Carlsbad,
142 CA, USA) were prepared in methanol and PBS, respectively, at 100 μM and 20 μL were
143 distributed to each well for a final concentration of 10 μM. Hydrogen peroxide (4M)
144 amphotericin B, and itraconazole were included as a control. The drug treatment solutions
145 were evaluated at the respective MIC, as published by Sá et al. (26), in a final volume of
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146 200 μL. The plates were incubated at 37ºC in the dark. After 1 hour, the fluorescence was o
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147 measured with a fluorometer (Synergy 2 SL Luminescence Microplate Reader, Biotek) lo
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148 using excitation 485 nm and emission wavelengths of 530 nm (30). d

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149 Checkerboard assay. Eight serial 2-fold dilutions of thiazoles compounds (0 - 16 μg/mL) om

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150 and glutathione (0 - 500 μg/mL), diamide (2.65 – 170 μg/mL), menadione (15.6 – 1000 t
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151 μg/mL), hydrogen peroxide (0.53 – 34 μg/mL) and parasulphonatephenyl porphyrinato /a
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152 ferrate III (FeTPPS, 3.18 – 204 μg/mL) were arrayed in serial concentrations. The .a
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153 checkerboard was prepared in a microtiter plate for multiple combinations of two .o
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154 molecules. Each row (x-axis) in the plate contained the -fold serial diluted concentration as /
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155 the thiazoles compounds. Similarly, each column (y-axis) in the plate contained 2-fold A
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156 serially diluted concentrations of the oxide stress agents. The concentration of the drug il 1
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157 combination at which growth was completely inhibited was taken as the effective MIC for 0
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158 the combination. Inocula suspension was performed as described by Sá et al. (26) and b
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159 100 µL of inocula was added to each well and cultured for 48-72 h. Fractional inhibitory u
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160 concentrations (FICs) were calculated as the MIC of the combination of drugs divided by
161 the MIC of drug alone. The same calculation was performed for all experimental
162 compounds. The FIC index (FICI) was calculated by adding both FICs, and this was
163 interpreted in the following manner: an effect was classified as synergistic when its value
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164 was ≤0.5, as no interaction when the value fell within the 0.5 to 4.0 range, and as
165 antagonistic when its value was >4.0 (31).
166 Erythrocyte toxicity assay. Erythrocyte toxicity assay was performed as described
167 previously by Evans et al. (32) with modifications. We evaluated the compounds for
168 hemolytic effects using red blood cells. Human erythrocytes (Rockland Immunochemicals,
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169 Pottstown, PA, USA) cells were diluted to 2% in PBS and 190 µL aliquots were added to a o
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170 96-well plate. An aliquot of the thiazole compounds, 10 µL, in PBS were added to the lo
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171 wells with red blood cells. The final concentration of investigational compounds in the d

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172 wells ranged from 0.125 to 64 µg/mL. As a positive control, Triton X-100 was included and om

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173 PBS was used as a negative control and a control with DMSO was also performed. The t
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174 plate was incubated at 37°C for 1 h and then centrifuged at 500 x g for 5 minutes to pellet /a
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175 the intact erythrocytes. 100 μL of the supernatant from each well was transferred to a new .a
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176 96-well plate. Percentage of hemolysis was determined by measuring absorbance at 540 .o
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177 nm (Bio-Rad Benchmark Plus spectrometer, Bio-Rad Laboratories, USA) and then /
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178 normalizing the mean absorbance value to the mean absorbance of the 1% Triton X-100 A
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179 treated samples, which represented 100% hemolysis. Two independent experiments were il 1
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180 performed in triplicate. 0
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181 Statistical analysis b
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182 Statistical analysis was performed using GraphPad Prism statistical software 5.0 u
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183 (GraphPad Software, Inc., San Diego, CA, USA). A 2-way ANOVA using Bonferroni post
184 test was used to statistically compare the differences among groups and a 95% CI was
185 considered in all experiments.
186
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187 RESULTS
188 In the present work we found that our group of investigational thiazole compounds
189 had no action on the fungal membrane or cell wall (Supplementary data). With evidence
190 that the thiazole compounds can inhibit mechanisms found within multiple fungi, we
191 endeavored to define the genetic targets of the compounds. Screening of a C. neoformans
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192 var. grubii H99 mutants library described by Liu et al. (29) for reduced sensitivity to our o
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193 investigational compounds revealed that mutants for the enzymes L-aminoadipate- lo
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194 semialdehyde dehydrogenase, sarcosine oxidase, and Cu-oxidase were resistant to thiazoles d

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195 (Table 2). Among these strains the Cu-oxidase mutant was the most resistant to thiazoles, om

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196 mainly to 1, 2, and 4, generating MICs superior to 8X the MIC value, when compared with t
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197 their wild type (WT). /a
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198 To evaluate if these thiazoles could affect any cellular antioxidant system, we tested .a
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199 the affect of the thiazole compounds against the thioredoxin system, an antioxidant pathway .o
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200 that did was not evaluated from our screen. We investigated the existence of interactions /
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201 between these compounds and glutathione, which in C. neoformans (KN99α) can function A
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202 as an antioxidant and complements the thioredoxin system, an antioxidant mechanism of il 1
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203 fungi. If the thioredoxin system were involved we would expect glutathione to act as an 0
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204 antagonist to the thiazole compounds. However, the MIC was not changed by the presence b
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205 of glutathione and FIC values were considered indifferent. u
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206 Based on the grouping of oxidase mutants that exhibited resistance to the
207 compounds, we quantified ROS and RNS of cells exposed to thiazoles to determine if they
208 act through changes to the antioxidant system of C. gattii L27/01 (= C. deuterogattii) and
209 C. neoformans ATCC 24067 (=C. deneoformans). Interestingly, we observed a significant
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210 increase in the amount of intracellular ROS after 1 hour of treatment with all the thiazole
211 compounds, but there was only a trend increase in RNS production (Figure 2a, b).
212 To better define the antioxidant systems potentially targeted by the thiazole
213 compounds, we performed a checkerboard assay with 1, 2, 3 and 4 combined with three
214 oxide stressors and one peroxinitrite sequestrant. We did not observe synergy between our
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215 investigational compounds and diamide, a thiol oxidizing agent, or hydrogen peroxide. o
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216 However, we found that menadione demonstrated synergistic interaction with thiazoles, lo
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217 since the FIC values were lower than 0.5 (Table 3). This interaction suggests that the d

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218 reductase system target of the thiazoles is related to the superoxide’s production. In om

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219 addition, there was an antagonistic interaction among thiazoles and FeTPPS, a highly t
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220 reactive nitrogen species, with FIC superior to 4.0 (Table 4). /a
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221 Systemic infections of cryptococcosis are treated via intravenous delivery of .a
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222 antifungal compounds. Thus, we examined if our group of investigational compounds could .o
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223 potentially be delivered into the bloodstream without initiating hemolytic effect. We tested /
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224 serial dilutions of 1, 2, 3, and 4 with 2% human erythrocytes and found less than 1% A
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225 hemolysis (Figure 3). The LC50 of the compounds against erythrocytes is higher than 64 il 1
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226 µg/mL. 0
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