The celecoxib derivative AR-12 has broad spectrum antifungal activity in vitro and improves the

49 Pages·2016·0.99 MB·English

AAC Accepted Manuscript Posted Online 19 September 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.01061-16
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 The celecoxib derivative AR-12 has broad spectrum antifungal activity in vitro and improves the
2 activity of fluconazole in a murine model of cryptococcosis
3 Running Title: Antifungal activity of AR-12
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5 Kristy Koselny1, Julianne Green1, Louis DiDone1, Justin P. Halterman1, Annette W. Fothergill3, o
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6 Nathan P. Wiederhold3, Thomas F. Patterson4,5, Melanie T. Cushion6, Chad Rappelye7, Melanie n
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7 Wellington1, and Damian J. Krysan1,2 d
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9 Departments of Pediatrics1 and Microbiology/Immunology2, University of Rochester School of ttp
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10 Medicine and Dentistry, Rochester, NY 14642. Department of Pathology3, University of Texas a
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11 Health Science Center at San Antonio, San Antonio, TX 78229, Department of Medicine4, .a
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12 University of Texas Health Science Center at San Antonio, and South Texas Veterans Health .
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13 Care System5, San Antonio, TX 78229, Department of Internal Medicine, University of Cincinnati /

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14 College of Medicine6, Department of Microbiology7, The Ohio State University, Columbus, OH,
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15 43210 il
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16 Corresponding Author: 0
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17 Damian J. Krysan y

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18 Department of Pediatrics and Microbiology/Immunology, University of Rochester, Box 850, s
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19 Elmwood Ave, Rochester NY, 14642.
20 Email: [email protected]
21 Tele: 585-275-5944; FAX: [phone]
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22 Abstract
23 Only one new class of antifungal drugs has been introduced into clinical practice in the last thirty
24 years and, thus, the identification of small molecules with novel mechanisms of action is an
25 important goal of current anti-infective research. Here, we describe the characterization of the
26 spectrum of in vitro activity and in vivo activity of AR-12, a celecoxib-derivative which has been
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27 tested in a Phase I clinical trial as an anti-cancer agent. AR-12 inhibits fungal acetyl CoA o
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28 synthetase in vitro and is fungicidal at concentrations similar to those achieved in human lo
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29 plasma. AR-12 has a broad spectrum of activity including active against yeasts (e.g., C. e
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30 albicans, non-albicans Candida spp., C. neoformans); molds (e.g., Fusarium, Mucor), and r
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31 dimorphic fungi (Blastomyces, Histoplasma, and Coccidioides) with minimum inhibitory h
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32 concentrations of 2-4 µg/mL. AR-12 is also active against azole- and echinocandin-resistant :
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33 Candida isolates and sub-inhibitory AR-12 concentrations increase susceptibility of fluconazole- c
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34 and echinocandin-resistant Candida isolates. Finally, AR-12 also increases the activity of s
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35 fluconazole in a murine model of cryptococcosis. Taken together, these data indicate that AR- r
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36 12 represents a promising class of small molecules with broad spectrum antifungal activity. o
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37 r
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44 Introduction
45 The treatment of life-threatening invasive fungal infections (IFIs) remains a significant challenge
46 to modern medicine (1, 2). Two primary factors contribute to the difficult nature of IFI therapy.
47 First, most IFIs affect people with compromised immune function and, therefore, IFI patients are
48 more reliant on the efficacy of the antifungal drug than immunocompetent patients. Second, the
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49 development of effective antifungal drugs is difficult because many fundamental biological o
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50 processes are highly conserved between fungi and humans (3). Consequently, identifying lo
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51 molecules that kill the pathogen and spare the host is very challenging. Currently, only three e
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52 primary classes of antifungal drugs are in clinical use: 1) azole ergosterol biosynthesis inhibitors r
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53 (e.g., fluconazole (FLU)); 2) polyene ergosterol binding agents (e.g., amphotericin B); and 3) h
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54 echinocandin 1,3-β-D-glucan synthase inhibitors (e.g., caspofungin). The number of antifungal :
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55 drugs pales in comparison to the number of distinct classes of antimicrobial agents and, in fact, c
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56 there are currently more classes of anti-retroviral agents available for the treatment of HIV/AIDS s
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57 than classes of antifungal drugs (2). r
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58 The pace of antifungal drug development has been extremely slow. For example, the n

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59 most recent addition to the antifungal pharmacopeia, the echinocandins, was discovered in the r
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60 early 1970s and introduced into clinical practice in the early 2000s (3). Furthermore, no new ,
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61 drugs for the treatment of cryptococcal meningitis, one of the most important causes of 1
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62 infectious disease related deaths in HIV/AIDS patients (4), have been introduced into clinical y

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63 practice in over twenty years. In fact, the current gold standard therapy is based on drugs e
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64 (amphotericin B and 5-flucytosine) that are fifty-years old (5). In principle, the low numbers of
65 drugs and the slow pace of development would not be an issue if the current therapies were
66 highly effective. Unfortunately, mortality rates associated with IFIs range from 20%-100%
67 depending on the organism and the immune status of the host (1). Clearly the pace of new
68 antifungal drug development needs to increase to meet the current need.
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69 As part of a program to identify and re-purpose protein kinase inhibitors as potential
70 antifungal drugs, we found that the anti-cancer clinical candidate OSU-03012/AR-12 is active
71 against C. albicans and C. neoformans in vitro with minimum inhibitory concentration (MIC) of 4
72 µg/mL (6). In addition, we have shown that AR-12 is synergistic with FLU against C.
73 neoformans (7) and is active against C. albicans biofilms (6). AR-12 is reported to be an
74 inhibitor of the conserved protein kinase PDK1 in humans and other mammals (8). Reports D
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75 from multiple groups, including our own, indicate that PDK1 may not be the target for AR-12 (9, n
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76 10). Indeed, we have recently found that AR-12 inhibits acetyl coenzyme A synthetase (Acs) in d
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77 yeast and that this mode-of-action is an important mechanism for its antifungal activity (10). Acs
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78 generates acetyl CoA from acetate and CoASH in an ATP-dependent reaction (11). In most

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79 yeast, acetate represents an important carbon source for acetyl CoA generation (12) and, thus, tp
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80 Acs activity has been found to be essential in C. albicans (13) and required for virulence in C. a
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81 neoformans (14). In contrast, the enzyme that generates the vast majority of acetyl CoA in a
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82 humans is ATP-citrate lyase (15). Recently, a number of studies have shown that cancer cells .
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83 have increased dependence on acetate and that Acs activity correlates with outcomes in some /

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84 cancer types (16, 17, 18). Based on these findings, Acs has been proposed as a possible anti- A
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85 cancer target (16, 19). We propose that these same considerations indicate that Acs may also il
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86 represent a potential antifungal target. 2
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87 AR-12 is derived from the cyclo-oxygenase 2 (Cox2) inhibitor celecoxib and has been b
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88 evaluated in phase I clinical trials as an anti-cancer agent (8, 20). As such, it has a number of
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89 favorable pharmacological properties. In addition, serum levels of AR-12 comparable to the t
90 vitro MIC for fungi were achieved in humans and mouse models (21). Encouraged by these
91 observations, we characterized the in vitro and in vivo antifungal activity of AR-12.
92
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93 MATERIALS AND METHODS
94 Strains and media. The clinical strains used for characterizing the activity of AR-12 against
95 pathogenic fungi were from the collection at the Fungus Testing Laboratory, University of Texas
96 Health Center at San Antonio. C. albicans strain SC5314 was obtained from Gus Haidaris
97 (Rochester) and Cryptococcus neoformans strain H99 was a gift of Joseph Heitman (Duke). C.
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98 albicans clinical isolates TWO7229, TWO7230, TWO7241, and TWO7243 were obtained from o
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99 Ted White (UMKC). C. albicans clinical isolates (10B1A3A and 11A8A2A) and SC5314 lo
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100 derivatives containing gain-of-function alleles (mrr1P683S and tac1G980E) were obtained from P. e
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101 David Rogers (Tennessee). C. albicans strains NC1010 and NCO-788 as well as C. glabrata r
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102 strains NC1, NC102, and NC999 were obtained from Neil Clancy and Ryan Shields (Pittsburgh). h
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103 SN250 and the mrr1Δ/Δ and tac1Δ/Δ derivatives were obtained from the Fungal Genetics Stock :
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104 Center. Yeast media was prepared using standard recipes. All yeast incubations were done at c
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105 30oC unless otherwise indicated. s
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106 g
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107 In vitro antifungal susceptibility assays. MIC values were determined using standard CLSI A
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108 methods except for those noted for H. capsulatum which was also performed using a recently il
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109 published protocol (22). Antifungal susceptibility testing was performed in the Fungus Testing 0
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110 Laboratory by the CLSI M27-A3 and M38-A2 methods for yeast and molds, respectively. The
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111 interaction of AR-12 with fluconazole was characterized by checkerboard assays using CLSI g
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112 M27-A3 methods. The fractional inhibitory concentration index was calculated as described s
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113 (23). Pneumocystis susceptibility testing was performed as previously described (13).
114
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115 Quantitative RT-PCR analysis of efflux pump expression. The strains were grown overnight
116 in YPD at 30oC and back diluted to 0.1 OD in YPD. The cultures were treated with sub-
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117 inhibitory AR-12 (4µg/mL) in DMSO (1%) or DMSO alone and incubated for 3 hr at 30 oC. The
118 cells were harvested by centrifugation and RNA isolated using Ambion Ribopure yeast kit. The
119 RNA was processed for SYBR Green RT-PCR using the iScript kit. Primers for the targets and
120 conditions for RT-PCR were exactly as previously reported (25). Expression was internally D
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121 normalized to ACT1 and compared to other strains or conditions using ΔΔC method. n
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122 e
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123 Microscopy. For assessment of the effect of AR-12 on mitochondrial activity, S. cerevisiae m

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124 BY4741 cells were grown to log phase, treated with AR-12 at concentrations indicated in text for t
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125 4 hours and harvested. The mitochondria were visualized with Mito-Tracker Red (Molecular /a
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126 Probes) following previously described protocols (26). Lipid droplet characterization was .
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127 performed using Nile Red staining as described (27). Chitin staining was performed with
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128 Calcofluor white as described in (28). Images were collected with a Nikon ES80 epi- g
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129 fluorescence microscope equipped with a CoolSnap CCD camera using NIS-Elements Software n

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130 using constant exposure settings and processed equivalently in PhotoShop. The percentage of r
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131 cells showing clearly staining mitochondria was determined by counting at least 100 cells per ,
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132 replicate for two independent experiments. Propidium iodide staining was performed as 1
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133 previously described (29). y

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134 s
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135 Glucose-induced acidification of culture medium by S. cerevisiae. Following methods
136 described by Soteropoulos et al. (30), logarithmic stage S. cerevisiae BY4741 cells were
137 harvested, suspended in 0.1 M KCl, and incubated at 30oC for 1 hr to starve the cells of
138 glucose. The suspensions were then stored overnight at 4oC at an OD of 2.3. The reactions
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139 contained 20 µL cell suspension in 155 µL of buffer (0.1 M KCl, 50 µg/mL bromophenol blue, pH
140 5) with either 1% DMSO alone or the indicated amount of AR-12 in 1% DMSO. Medium
141 acidification was initiated by the addition of 20 µL of 20% glucose (wt/volume). The progress of
142 the reaction was followed by measurement of A on a plate reader at 30 min intervals over a 4
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143 hr time-course. The reduction in A for the untreated cells over this time course was essentially
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144 identical to that reported by Soteropoulos et al. (30). D
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145 lo
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146 Isolation and analysis of AR-12 resistant mutants. S. cerevisiae strain BY4741 was d

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147 passaged in culture flasks containing YP+2% acetate starting at 1 µg/mL (0.125X MIC; MIC = 4 m

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148 µg/mL) and increasing 2-fold to 64 µg/mL. The flasks were incubated until saturated. Samples t
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149 harvested and used to inoculate a flask with a 2-fold higher concentration in AR-12. At /a
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150 concentrations above MIC (16, 32, and 64 µg/mL), a dilution of the culture was plated on YPD .
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151 and single colonies were isolated. The colonies were passaged twice on non-selective YPD
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152 plates. Colonies from the passaged plates were incubated in micotiter plates containing the AR- g
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153 12 concentration from which they were isolated and compared to the parental strain. Resistant n

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154 isolates were then compared to the parental strain by growth curve analysis in the presence and r
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155 absence of AR-12. Three isolates showed reproducible, stable resistant to AR-12 and were ,
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156 analyzed by whole-genome re-sequencing. Sequenced reads were cleaned according to a 1
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157 rigorous pre-processing workflow (Trimmomatic-0.32) before mapping them to the S. cerevisiae y

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158 genome (SGD) (EF4) with SHRiMP2.2.3 ( http://compbio.cs.toronto.edu/shrimp/ ). SAMtools-1.0 e
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159 and BCFtools-1.0 were used to perform the SNP and INDEL calling. Then a filter for low
160 coverage (10x) and low quality (20) was applied and marked in filter column either "PASS" or
161 "LowQual". Non-synonymous mutations in confirmed ORFs were identified using UCSC
162 Genome Bioinformatics website (http://genome.ucsc.edu) and SGD
163 (http://www.yeastgenome.org/). The relative susceptibility of the mutants and reference strains
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164 to AR-12 and fluconazole were determined by incubating the strains in the presence of AR-12
165 overnight and then plating a 10-fold dilution series on non-selective YPD media to compare the
166 number of viable cells in each culture.
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168 Mouse model of disseminated cryptococcosis. Male AJ/Cr mice (20-25 gm) were D
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169 purchased from the Frederick National Laboratory for Cancer Research (NCI, Frederick, MD).
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170 Animals were housed in the University of Rochester Medical Center vivarium and allowed food a
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171 ad libitum. On day 0, mice were inoculated via lateral tail vein injection with 4.5 x 104 cfu/animal d

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172 of C. neoformans H99 in PBS (100 µL). Beginning 24 hours after inoculation and continuing m

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173 daily for days 2-6, mice were sham treated (n =8); treated with FLU by intra-peritoneal injection t
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174 (10 mg/kg in PBS; n = 8); treated with AR-12 by oral gavage (100 mg/kg suspended PBS with /a
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175 0.5% methylcellulose/0.1%Tween 80: n =8) and treated with both FLU and AR-12 (n =8). On .
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176 day 7, all mice were euthanized after which the brains were removed and homogenized in YPD
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177 (2 mL). Serial dilutions of the homogenates were inoculated on YPD agar plates containing g
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178 vancomycin (10 µg/mL) and gentamicin (100 µg/mL). The colony forming units per gram of n

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179 brain tissue was calculated, transformed into log units, and differences between groups r
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180 analyzed by ANOVA; statistical significance was set at p < 0.05 (SigmaPlot Software). The ,
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181 experiment was performed twice with similar results for each replicate. 1
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183 t
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186
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187 Results
188 AR-12 has broad spectrum antifungal activity against pathogenic yeast, molds,
189 dimorphic fungi, and pneumocystis.
190 We initially identified AR-12 (Fig. 1) as an antifungal molecule by screening a series of
191 molecules with activity against human PDK1 (6); as discussed above, subsequent studies have D
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192 led to the conclusion that the biological activity of AR-12 is unlikely to be due to PDK1 inhibition.
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193 AR-12 is fungicidal against both C. albicans and C. neoformans (MIC/MFC 4 µg/mL for both a
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194 organisms), synergistic with FLU toward C. neoformans; and has activity toward C. albicans d

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195 biofilms within 2-fold of its MIC toward planktonic cells (6, 7). The initial studies of AR-12 m

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196 antifungal activity were limited to the standard laboratory strains of C. albicans (SC5314) and C. t
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197 neoformans (H99, K99, JEC21). To further confirm and validate this activity, we tested a set of /a
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198 clinical isolates of C. albicans, non-albicans Candida spp., and C. neoformans (Table 1). .
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199 Consistent with previous results, AR-12 is active against multiple clinical isolates of C. albicans
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200 (MIC 4 µg/mL), including those with decreased susceptibility to FLU. AR-12 is similarly active g
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201 against non-albicans Candida spp. (Table 1), including the relatively FLU-resistant species C. n

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202 glabrata and C. krusei as well as C. parapsilosis, C. dublineinsis and C. tropicalis. Finally, r
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203 clinical isolates of C. neoformans var. grubii were as susceptible as the standard laboratory ,
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204 strains. Consequently, AR-12 has consistent activity against clinical isolates of pathogenic 1
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205 yeast. The MIC for these strains (4 µg/mL) is similar to AR-12 serum concentrations (8 µM or y

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206 3.7 µg/mL) observed in Phase I clinical trials (S. Proniuk, personal communication). e
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207 One of the most pressing un-met clinical needs in medical mycology is the development
208 of agents with activity against difficult to treat molds that cause aspergillosis,
209 hyalohyphomycoses, phaeohyphomycoses, and mucormycosis. Therefore, we tested the
210 activity of AR-12 against a panel of medically relevant molds (Table 1). AR-12 is active against
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211 A. fumigatus and this activity is further characterized in a companion article. Mucomycosis has
212 emerged as an increasingly prevalent infection, particularly within severely
213 immunocompromised patients (31). AR-12 has activity against the most common cause of
214 mucormycosis, R. oryzae, as well as two out of four isolates of the less commonly isolated
215 species Apophysomyces. Except for the two isolates noted, the AR-12 MIC against was 4
216 µg/mL, which is lower than that observed for posaconazole. D
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217 AR-12 is also active against molds that cause phaeohyphomycosis (Table 1) including F. lo
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218 solani and F. oxysporum; S. apiospermum; Paecilomyces, and Lomentospora prolificans. e
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219 Notably, AR-12 is more active against species that cause fusariosis than voriconazole, the r
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220 recommended therapy for this difficult to treat mold infection. It is also more active than h
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221 voriconazole against L. prolificans but less active against S. apiospermum and Paecilomyces. :
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222 We also tested the activity of AR-12 against three medically important dimorphic fungi. c
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223 Consistent with the other organisms, AR-12 showed activity in the 2-4 µg/mL range against H. s
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224 capsulatum, B. dermatitidis, Coccidioides spp.. The activity against H. capsulatum varied r
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225 somewhat with the methodology; the MIC obtained with a recently developed method optimized o
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226 for the yeast phase rather than against hyphae was higher with consistent values of 4 µg/mL p
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227 (22). il 5
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228 Finally, we determined the activity of AR-12 against Pneumocystis, an opportunistic 1
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229 fungal pathogen not susceptible to standard antifungal agents. Mammals are infected by host- y

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230 specific species of Pneumocystis which are not culturable in vitro under normal laboratory e
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231 conditions. Therefore, AR-12 was tested against rat (P. carinii) and mouse (P. murina) specific
232 species using an ATP-based viability assay (13). AR-12 showed moderate activity against both
233 species with 72-hour IC of 4.8 µg/mL for P. carinii and 1.8 µg/mL for P. murina. IC values for
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234 pentamidine, an agent used for treatment and prophylaxis, are typically less than 1µg/mL (24).
235 AR-12 has a remarkably broad spectrum of antifungal activity at concentrations that are similar
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