AAC Accepted Manuscript Posted Online 30 April 2018 Antimicrob. Agents Chemother. doi:10.1128/AAC.00504-18 Copyright © 2018 American Society for Microbiology. All Rights Reserved. 1 Title: Commonly used oncology drugs decrease antifungal effectiveness against Candida and 2 Aspergillus species 3 4 Authors: Arielle Butts1, Parker Reitler2, Wenbo Ge1, Jarrod R. Fortwendel1, and Glen E. 5 Palmer1# D o 6 Running Title: Drug induced antifungal resistance w n 7 lo a d e 8 d f r 9 Affiliations: 1Department of Clinical Pharmacy and Translational Science, College of Pharmacy, om h 10 University of Tennessee Health Sciences Center, Memphis, USA. 2Department of Molecular tt p : / / 11 Immunology and Biochemistry, College of Graduate Health Sciences, University of Tennessee a a c . 12 Health Sciences Center, Memphis, USA. a s m 13 .o r g 14 o/ n 15 # Corresponding author. Mailing Address: University of Tennessee Health Sciences Center, A p r 16 College of Pharmacy, Department of Clinical Pharmacy, 881 Madison Avenue, Memphis, il 5 , 2 17 Tennessee, 38163. 0 1 9 18 Phone: (901) 448-3744. b y g 19 Fax: (901) 448-7053. u e s t 20 Email: [email protected]. 21 1 22 Abstract. 23 The incidence of invasive fungal infections has risen significantly in recent decades as medical 24 interventions have become increasingly aggressive. These infections are extremely difficult to 25 treat due to the extremely limited repertoire of systemic antifungals, the development of drug 26 resistance, and the extent of to which the patient’s immune function is compromised. Even when D o w 27 the appropriate antifungal therapies are administered in a timely fashion, treatment failure is n lo a 28 common, frequently even in the absence of in vitro microbial resistance. In this study, we d e d 29 screened a small collection of FDA approved oncolytic agents for compounds that impact the fr o m 30 efficacy of the two most widely used classes of system antifungals against Candida albicans, h t t p 31 Candida glabrata, and Aspergillus fumigatus. We have identified several drugs that enhance : / / a a 32 fungal growth in the presence of the azole antifungals and examine the potential that these drugs c . a s 33 directly affect fungal fitness, specifically antifungal susceptibility, and may be contributing to m . o 34 clinical treatment failure. rg / o n A 35 Keywords. Antagonism, induced resistance, antifungal treatment failure, azoles, echinocandins p r il 5 , 36 Introduction. 2 0 1 37 The global burden of invasive fungal infections (IFIs) has increased dramatically as the 9 b y 38 population of susceptible individuals continues to expand (1). Worryingly, the mortality rate for g u e 39 many IFIs exceeds 50% despite the provision of appropriate antifungal agents. While the s t 40 increasing incidence of antifungal drug resistance undoubtedly contributes to the frequency of 41 treatment failure (2, 3), in vitro resistance is only observed in about one third of such cases. 42 While a variety of factors have been speculated to account for the remaining non-responsive 43 patients including inadequate drug distribution or severity of immune dysfunction, little evidence 2 44 has been provided in support of these arguments. We considered an additional explanation - the 45 influence of other medications on the fungal pathogen itself. This is especially pertinent given 46 that individuals at greatest risk of developing IFIs are usually receiving a multitude of drugs to 47 treat a variety of underlying conditions (4). Furthermore, as eukaryotes, human and fungal cells 48 share the same basic biology and signaling pathways. Accordingly, many drugs that induce a D o 49 physiological response in humans are likely to induce a response in fungi. Yet the influence of w n 50 most medications upon fungal physiology, antifungal susceptibility, a patient’s response to lo a d e 51 antifungal therapy, and in a broader sense the outcome of infection, remains largely unknown. d f r o 52 Several approved drugs are known to enhance the efficacy of existing antifungal medications, m h 53 and may therefore provide a basis for adjunctive therapies (5). However, to date, there has been tt p : / / 54 no systematic attempt to identify approved medications that promote survival of infectious fungi a a c . 55 in the presence of antifungal drugs and may therefore undermine their clinical efficacy. Thus, a s m 56 while drug-drug interactions are a serious concern from the perspective of patient toxicity, the .o r g / 57 consequences of similar interactions on the fungal pathogen itself has not been widely o n A 58 appreciated. The purpose of this study was to determine the effect of approved oncology drugs p r 59 on the efficacy of the two most important classes of antifungal pharmacotherapies - the azoles il 5 , 2 60 and the echinocandins. In so doing, we focused on three of the most prevalent fungal pathogens 0 1 9 61 within this patient population, Candida albicans, Candida glabrata, and Aspergillus fumigatus b y g 62 (6, 7). u e s t 63 Results. 64 We screened a library of oncology drugs to identify any that interfere with the antifungal activity 65 of the azoles or the echinocandins, under conditions that closely mimic those used in the CLSI 66 protocol for determining minimum inhibitory concentrations (MICs). Fungal cells were diluted 3 67 into RPMI medium with supra-inhibitory concentrations of the selected antifungal and dispensed 68 into the wells of 96-well plates, each containing a single test compound to a final concentration 69 of 5 µM. Oncology drugs that increased fungal growth at least 2-fold versus the antifungal drug 70 alone control were called hits. Of the 129 compounds in the library, twenty-one were identified 71 as hits in at least one screen, indicating that this type of interaction may be far more common D o 72 than originally anticipated. w n 73 Candida albicans. Eight compounds were identified as enhancing C. albicans growth in the lo a d e 74 presence of 1 µM fluconazole (~8X MIC) (Table 1 and Figure 1A). Strikingly, all but one of d f r o 75 these drugs belong to one of two classes – specifically topoisomerase or kinase inhibitors. A m h 76 representative of each class causing the greatest restoration of fungal growth in the presence of tt p : / / 77 fluconazole, idarubicin and ceritinib, as well as the unrelated microtubule inhibitor, cabazitaxel, a a c . 78 were selected for follow-up analysis. Checkerboard assays were conducted to confirm and a s m 79 determine the extent of antifungal antagonism, as well as determine the effective concentration .o r g / 80 range of each hit (Figure 2). Certinib had a paradoxical effect with a dose-dependent increase in o n A 81 antagonism up to 0.625 µM at which point a 16-fold increase in resistance was observed, but at p r 82 higher concentrations it enhanced fluconazole’s antifungal activity. Idarubicin exhibited a dose- il 5 , 2 83 dependent increase in antifungal antagonism at concentrations ≥78 nM, inducing a 16-fold 0 1 9 84 increase in fluconazole resistance at 5 µM. Cabazitaxel also antagonized fluconazole’s activity at b y g 85 concentrations ≥0.625 µM with 4-fold resistance observed at 2.5 µM. To determine if these u e s t 86 interactions were specific to fluconazole, we performed checkerboard assays with two additional 87 azole antifungals, itraconazole and voriconazole. While the extent of induced resistance and the 88 specific concentrations at which the effects were observed varied, all three oncology agents 89 tested also reduced the effectiveness of itraconazole and voriconazole (Figure 3). Finally, we 4 90 tested if a combination of these three agents would act in concert to further elevate fluconazole 91 resistance, however, no additive effects were observed upon fluconazole resistance in the 92 selected C. albicans strain (data not shown). Nonetheless, it remains possible that specific 93 combinations of the other antagonistic oncology agents may induce resistance of greater 94 magnitude than when provided alone. Using the same screening strategy, we did not identify any D o 95 oncology agents that were able to rescue C. albicans growth in the presence of 500 nM w n 96 caspofungin (~8X MIC), indicating that the observed interactions are antifungal specific. lo a d e 97 Candida glabrata. Surprisingly, a total of eleven oncology drugs were identified as facilitating d f r o 98 C. glabrata growth in the presence of 100 µM fluconazole (~8X MIC), including one m h 99 topoisomerase inhibitor and one kinase inhibitor (Table 1 and Figure 1B). While the specific tt p : / / 100 drugs inducing fluconazole resistance in C. glabrata were distinct from those identified for C. a a c . 101 albicans, the common drug classes identified suggest that the underlying mechanisms are likely a s m 102 similar for many of the antagonistic interactions observed for either species. Additionally, .o r g / 103 several steroid-like compounds, abiraterone, exemestane, and megestrol were identified as o n A 104 antagonizing fluconazole’s activity upon C. glabrata and selected for follow-up (Figure 4). We p r 105 were not able to confirm the antagonistic interaction with megestrol, indicating it was a false il 5 , 2 106 positive. However, exemestane induced a modest 2-fold increase in fluconazole MIC at 0 1 9 107 concentrations of 2.5-5 µM, while abiraterone produced a 4-fold increase in MIC at b y g 108 concentrations as low as 0.156 µM. Tretinoin was the only agent identified in the caspofungin u e s t 109 antagonism screen with C. glabrata, however, this interaction was not confirmed in follow-up 110 experiments. 111 Aspergillus fumigatus. Three antimetabolite compounds, floxuridine, fluorouracil, and 112 thioguanine, were identified as enabling A. fumigatus growth in the presence of 2 µg/mL 5 113 voriconazole (~4X MIC). Floxuridine is a prodrug that is rapidly converted into fluorouracil, it is 114 therefore likely these agents act via the same mechanism. Both fluorouracil and thioguanine were 115 tested in checkerboard assays and confirmed to enhance A. fumigatus growth in the presence of 116 voriconazole in a dose dependent manner (Figure 5). While fluorouracil treatment enhanced 117 fungal growth starting at 39 nM, it only caused a 2-fold increase in the voriconazole MIC at the D o 118 highest concentration tested of 5 µM. Thioguanine enhanced fungal growth in the presence of w n 119 voriconazole at concentrations as low as 19 nM, however it did not shift the MIC at any lo a d e 120 concentration tested. When this collection was screened for compounds that support A. d f r o 121 fumigatus growth in the presence of 1 µM caspofungin (~4X minimum effective concentration), m h 122 no hits were identified, further supporting that the observed interactions are antifungal specific. tt p : / / 123 Discussion. a a c . 124 While there have been reports of antagonism occurring with specific combinations of antifungal a s m 125 drugs (8, 9) including between flucytosine and fluconazole against C. glabrata (10), this is, to .o r g / 126 the best of our knowledge, the first study to systematically assess the influence of approved o n A 127 medications upon the activity of antifungal drugs. Our results indicate that this phenomenon may p r 128 be more common than previously appreciated and may contribute to currently unexplained il 5 , 2 129 clinical treatment failure, especially in specific patient cohorts. The number of oncology drugs 0 1 9 130 that negatively impact the antifungal activity of azoles was particularly surprising, especially b y g 131 considering the dearth of interactions with echinocandins. There are likely several factors u e s t 132 contributing to this disparity that relate to the target enzymes characteristics as well as the 133 physiological consequences of their inhibition. For example, fluconazole is fungistatic against 134 Candida sp., and thus fungal cells have an opportunity to mount a drug-induced adaptive 135 response that enables growth to resume. In contrast, the echinocandins are fungicidal, which may 6 136 restrict the opportunity to mount an adaptive response of sufficient magnitude to promote 137 survival and proliferation. The distinct cellular location of the target enzymes may also be 138 pertinent. The azole target enzyme, lanosterol demethylase (Erg11p) is intracellular, and thus 139 mechanisms or responses resulting in decreased antifungal drug uptake, membrane permeability 140 or enhanced efflux can confer resistance. In contrast, the echinocandins target β1-3 glucan D o 141 synthase, which is exposed at the cell surface and therefore not affected by efflux or cell w n 142 permeability issues. lo a d e 143 These findings raise several questions that require urgent attention: First, are the antagonistic d f r o 144 interactions observed of clinical relevance or merely artefacts of in vitro culture? Moreover, can m h 145 these interactions help explain the large number of treatment failures occurring in patients with tt p : / / 146 IFIs that are not accounted for by heritable resistance of the infecting fungus? Investigation of a a c . 147 this question will necessitate determining if the antagonistic drugs are able to undermine a s m 148 antifungal efficacy at pharmacologically relevant concentrations, the use of appropriate animal .o r g / 149 models of infection, and an analysis of patient data. Initial studies indicate that all antagonistic o n A 150 agents confirmed herein exert an effect within an order of magnitude of reported plasma p r 151 concentrations, supporting the notion that these interactions may have clinical relevance il 5 , 2 152 [www.micromedexsolutions.com]. Second, aside from oncology related drugs, are there other 0 1 9 153 pharmacotherapies that interfere with antifungal efficacy? Third, what are the underlying b y g 154 mechanisms of the observed antagonism? Azole resistance in C. albicans can be conferred by u e s t 155 elevated expression of drug efflux pumps belonging to the major facilitator and ABC transporter 156 super families, as well as the target enzyme itself of the target enzyme, Erg11p (11). It is likely 157 that some of these agents are acting through induction of these mechanisms. For example, the 158 expression of several ergosterol biosynthetic genes has been shown to be responsive to various 7 159 steroids (12). In addition, the transcription factors that regulate efflux pump expression are 160 known to bind to and be activated by a variety of xenobiotics (13). For example, the antifungal 161 flucytosine, which is metabolized into fluorouracil, and has been shown to enhance efflux pump 162 expression in C. glabrata in a Pdr1p dependent manner (14). Some negative interactions may 163 stem from drug-induced activation of stress responses that promote fungal survival upon D o 164 antifungal challenge. It is also possible that some interactions result from direct chemical w n 165 interaction or reaction leading to inactivation of the antifungal drug. Future work should focus on lo a d e 166 determining which mechanisms predominate and to what extent that varies across and within the d f r o 167 classes of agents identified. Fourth, can drug-induced antifungal resistance act in concert with m h 168 genetically encoded mechanisms to exacerbate resistance and/or tolerance? The latter point is tt p : / / 169 highly important as clinically relevant levels of resistance to the azole antifungals in C. albicans a a c . 170 usually depends upon a combination of mechanisms (15). Fifth, do similar antagonistic drug a s m 171 interactions occur with additional infectious fungal species or, more broadly, with other .o r g / 172 pathogenic microbes including protozoan parasites and bacteria? While we may expect fewer o n A 173 pharmacotherapies to interact with prokaryotes due to the greater evolutionary divergence p r 174 between bacterial pathogens and their mammalian host, many protein classes are well conserved il 5 , 2 175 across all forms of life. Thus, the potential exists for drugs targeted at human proteins to engage 0 1 9 176 bacterial homologs, or by unrelated mechanisms alter bacterial physiology to promote survival in b y g 177 the presence of antibacterial agents. Improving our understanding of how widespread the u e s t 178 phenomenon of drug-induced antimicrobial resistance is, as well as identifying the underlying 179 mechanisms, will be crucial to optimize therapeutic selection and ultimately improve patient 180 outcomes. 181 Materials and Methods. 8 182 Strains. Candida albicans strain SC5314 (16), Candida glabrata strain ATCC2001 (17), and 183 Aspergillus fumigatus strain Af293 (18) were used throughout this study. 184 Antifungal susceptibility testing. All susceptibility testing was performed in accordance with 185 the Clinical Laboratory and Standards Institute (CLSI) broth microdilution protocols (M27-A3) 186 (19) using RPMI 1640 medium buffered with morpholinepropanesulfonic acid (MOPS) and pH D o 187 adjusted to 7.0, except where specifically noted otherwise. w n 188 Compound library. A collection of 129 FDA approved oncology agents was provided by the lo a d e 189 Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National d f r o 190 Cancer Institute, part of the National Institutes of Health (NIH). m h 191 Screening. The wells of the 96-well flat-bottom assay plates were seeded with 1 L of the 1 mM tt p : / / 192 stock solutions of each library compound in DMSO or DMSO alone. Approximately 1000 cells a a c . 193 of either Candida species from an overnight culture were added to each well in 199 L RPMI a s m . 194 1640 medium containing the indicated concentrations of fluconazole or caspofungin, and o r g / 195 incubated as described in the CLSI protocol. After 24 or 48 hours, cells were manually o n A 196 resuspended before OD600nm was measured using a microplate reader. For A. fumigatus, 20000 p r il 197 conidia were inoculated per ml of the RPMI medium and germinated at 37°C with shaking at 5 , 2 0 198 250 rpm for 4 hr, before the indicated antifungal drugs were added. Subsequently, 199 µL of the 1 9 b 199 culture was dispensed into the assay wells. Due to the limitations of OD600nm with filamentous y g u 200 fungi, all determinations were made by visual inspection. In all wells the final DMSO e s t 201 concentration was 0.55%. 202 203 Acknowledgements. 9 204 Research reported in this publication was supported by the National Institute of Allergy and 205 Infectious Diseases of the National Institutes of Health under Award Number R01AI099080 206 (awarded to GP). The content is solely the responsibility of the authors and does not necessarily 207 represent the official views of the National Institutes of Health. JRF and WG are supported by 208 NIH R01 AI106925 to JRF. The authors would also like to thank the National Cancer Institute D o 209 (NCI), part of the National Institutes of Health for providing the library of oncology related w n 210 drugs through the Open Chemical Repository Developmental Therapeutics Program. lo a d e 211 d f r o 212 Figure 1. Identification of oncology agents that induce in vitro fluconazole resistance. (A) C. m h 213 albicans strain SC5314 and (B) C. glabrata strain ATCC2001were grown in the presence of 1 tt p : / / 214 and 100 µM fluconazole respectively in RPMI medium supplemented with a final concentration a a c . 215 of 5 µM of each compound from the NCI oncology collection. After 24 hours incubation at a s m 216 35°C, growth was measured as OD600nm, and normalized to the fluconazole alone controls (red .o r g / 217 squares). A second set of no-drug control wells had DMSO solvent alone (green circles). o n A 218 p r 219 Figure 2. Oncology drugs reduce Candida albicans susceptibility to fluconazole. il 5 , 2 220 Checkerboard assays in RPMI were performed with C. albicans strain SC5314 across a range of 0 1 9 221 fluconazole doses and (A) certinib, (B) cabazitaxel, and (C) idarubicin concentrations. After 24 b y g 222 hours of incubation at 35°C, growth was quantified by OD , normalized to the untreated u 600nm e s t 223 control well, and presented as a heat map. 224 225 Figure 3. The antagonistic effect of several oncology agents is not fluconazole specific. 10