Pathogens and Disease ISSN 2049-632X

RESEARCH ARTICLE

High-content phenotypic screenings to identify inhibitors of Candida albicans biofilm formation and filamentation Christopher G. Pierce, Stephen. P. Saville & Jose L. Lopez-Ribot Department of Biology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, TX, USA

This paper describes a high-throughput screening method to identify compounds that inhibit biofilm formation and/or filamentation by the human fungal pathogen Candida albicans. Several molecules with good activity have been identified, but toxicity issues will have to be tackled before this will result in novel antifungal lead compounds.

Keywords Candida albicans; biofilms; filamentation; high-content screening; chemical library. Correspondence  L. Lopez-Ribot, Department of Biology, Jose The University of Texas at San Antonio, San Antonio, TX 78249, USA. Tel.: +1 210 458 7022 fax: +1 210 458 7023 e-mail: [email protected] Received 26 November 2013; revised 13 January 2014; accepted 11 February 2014. Final version published online 11 March 2014. doi:10.1111/2049-632X.12161

Abstract Candida species represent the main cause of opportunistic fungal infections worldwide, and Candida albicans remains the most common etiological agent of candidiasis, now the third to fourth most common nosocomial infection. These infections are typically associated with high morbidity and mortality, mainly due to the limited efficacy of current antifungal drugs. In C. albicans, morphogenetic conversions between yeast and filamentous forms and biofilm formation represent two important biological processes that are intimately associated with the biology of this fungus and also play important roles during the pathogenesis of candidiasis. We have performed cell-based phenotypic screens using three different chemical libraries from the National Cancer Institute’s Open Chemical Repository collection and identified several compounds with inhibitory activity against C. albicans biofilm formation and/or filamentation. These phenotype-based approaches represent a prosperous alternative to conventional genetics and genomics techniques to address experimentally challenging and complex biological phenomena, such as biofilm formation and filamentation, while at the same time opening new possibilities for the development of new antifungal agents.

Editor: Patrick van Dijck

Introduction Candida albicans is a common commensal of the human oral, vaginal and gastrointestinal tracts, where it normally causes little or no damage to the host; however, as opportunistic pathogenic fungus, it is fully capable of causing infections ranging from superficial to life-threatening systemic candidiasis in immunosuppressed or otherwise compromised individuals (Kadosh & Lopez-Ribot, 2013). Candidiasis is now the third to fourth most common nosocomial infection in hospitals (Pfaller & Diekema, 2007), with mortality rates up to 50% (Gudlaugsson et al., 2003; Pappas et al., 2003; Pfaller & Diekema, 2007). These fungal infections are generally associated with the formation of biofilms, which are structured microbial communities attached to a surface and encased in an exopolymeric matrix (Ramage et al., 2009). Biofilm formation occurs on both biological and inert surfaces and is of significant clinical importance because of the widespread use of medical implant devices, such as stents, shunts, prostheses, endo-

tracheal tubes, pacemakers, and various types of catheters (Kojic & Darouiche, 2004; Ramage et al., 2006). From a clinical point of view, C. albicans biofilms have increased resistance to commonly used antifungal agents such as fluconazole and amphotericin B as compared to their planktonic counterpart (Mathe & Van Dijck, 2013; Pierce et al., 2013). Currently, echinocandins and liposomal formulations of amphotericin B are the only antifungals shown to be effective in the treatment of C. albicans biofilm infections (Bachmann et al., 2002; Kuhn et al., 2002; Kucharikova et al., 2011, 2013). Structurally, fully mature C. albicans biofilms are a complex mixture of yeast, hyphae, and pseudohyphae (Nett & Andes, 2006), and the ability of the fungus to reversibly transition between these morphologies plays an important role in biofilm development (Baillie & Douglas, 1999; Ramage et al., 2002; Lopez-Ribot, 2005). In addition to its important role in biofilm formation, the phenotypic switch from yeast to filamentous forms, which can occur in response to various stimuli within the host, also represents

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

423

Screening for inhibitors of C. albicans biofilms

a major virulence factor during candidiasis (Saville et al., 2003; Kadosh & Lopez-Ribot, 2013). Here, we have conducted whole cell-based phenotypic screenings to identify compounds present in the National Cancer Institute’s Open Chemical Repository collection with inhibitory activity against C. albicans biofilm formation and filamentation.

Materials and methods Chemical libraries Chemical libraries were obtained from the National Cancer Institute and a total of 2293 compounds from three libraries (1) Natural Set, (2) Structural Diversity Set, and (3) Challenge Set, were screened. Detailed information on these libraries can be found at http://www.dtp.nci.nih.gov/ branches/dscb/repo_open.html. The compounds in the libraries are provided as 10 mM solutions in DMSO. An initial 1 : 100 dilution for each compound was prepared by pipetting 2 lL of this concentrated solution into 198 lL of PBS using the wells of presterilized, polystyrene, roundbottomed, 96-well microtiter plates (Corning Incorporated, Corning, NY) and stored as working stock solutions at 20 °C. Strains, media, and culture conditions The strains used in the present study were C. albicans wild-type strain SC5314 and C. albicans tet-NRG1 strain (Saville et al., 2003). Overnight cultures of the strains were grown by inoculating in 20 mL of yeast peptone dextrose [YPD; 1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose] liquid media in 150-mL flasks and incubating in an orbital shaker (150–180 r.p.m.) at 30 °C. Under these conditions, both strains of C. albicans grow as budding yeast. In the C. albicans tet-NRG1 strain, morphogenetic conversions can be regulated both in vitro and in vivo by the presence or absence of doxycycline (Saville et al., 2003). In the absence of doxycycline, high NRG1 expression levels block the yeast to hyphae transition. The presence of doxycycline inhibits the expression of the tet-NRG1 allele and filamentation occurs normally in response to appropriate external/environmental stimuli. Screening chemical libraries for effects on biofilm formation Candida albicans wild-type strain SC5314 was used to screen the library of small molecules for effects on biofilm formation. Biofilms were formed using the 96-well microtiter plate model previously described by our group (Ramage et al., 2001; Pierce et al., 2008). Briefly, cells were harvested from overnight YPD cultures and after washings they were resuspended in RPMI-1640 supplemented with L-glutamine (Cellgro) and buffered with 165 mM morpholinepropanesulfonic acid (MOPS) at a final concentration of 1.0 9 106 cells mL 1. Candida albicans biofilms were formed on commercially available presterilized, polystyrene, 424

C.G. Pierce et al.

flat-bottomed, 96-well microtiter plates (Corning Incorporated). To the first column of the plate, DMSO was added at a concentration of 0.05%, equivalent to the concentration in the wells containing compounds. This DMSO concentration is not toxic to Candida and does not affect biofilm formation as previously demonstrated in our laboratory (Ramage et al., 2001). Columns 2 through 11 of the microtiter plate contained 5 lL of previously diluted compounds. The final concentration at which the compounds were tested was 5 lM. The final column of the plate remained empty to serve as a background control when the OD was determined in a microtiter plate reader. The initial screening was carried out in duplicate and the plates were incubated at 37 °C for 24 h. Following biofilm formation, the wells were washed twice to remove nonadherent cells, visualized by light microscopy and processed using semi-quantitative colorimetric assay based on the reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetra-zolium-5-carboxanilide (XTT; Sigma) as previously described by our group (Ramage et al., 2001; Pierce et al., 2008). The OD of control biofilms formed (in the absence of compound) was arbitrarily set at 100% and the inhibitory effects of compounds were determined by the percent reduction in absorbance in relation to the controls. Data were calculated as percent biofilm inhibition relative to the average of the control wells. In initial biofilm screens, active molecules were identified as those showing a reduction of 50% or greater in duplicate wells as compared to the controls. Screening chemical libraries for effects on C. albicans filamentation The genetically engineered C. albicans tet-NRG1 strain was used to screen for inhibitors of filamentation. The screening was carried out in 96-well round-bottomed plates. For the initial screening, 10 lL of the previously diluted compounds was added to the wells of the microtiter plates. Overnight cultures (14 h) were grown in YPD liquid media, and 1 in 30 dilutions were made in fresh YPD liquid media and YPD containing doxycycline. The initial screening was carried out in duplicate, and plates were incubated at 37 °C and examined at 2, 4, and 24 h, both macroscopically (if cells remain in the yeast form, they fall to the bottom of the round-bottomed wells forming a ring that is easily identifiable by the naked eye looking at the bottom of the plates) and microscopically. Confirmation of hits and determination of potency using dose–response assays Compounds identified as initial hits in the screening of inhibitors of C. albicans wild-type biofilms were confirmed via dose–response assays. These assays used the same 96-well flat-bottomed plate model for biofilm formation used in the initial screening, except the compounds were screened in serial twofold dilutions with concentrations ranging from 40 to 0.078 lM. The compounds were serially diluted in 50 lL of RPMI directly in columns of the microtiter plates, with appropriate positive and negative controls. Each assay was performed in duplicate. After incubation, plates

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

C.G. Pierce et al.

were washed twice and processed using the XTT reduction assay. From the dose–response assays, the IC50 value, defined as the concentration of each compound leading to 50% inhibition of biofilm formation, was calculated using GRAPHPAD PRISM software. Compounds considered as initial hits in the primary screening of inhibitors of C. albicans filamentation in the tet-NRG1 strain were confirmed using dose–response assays. The same 96-well round-bottomed microtiter plate model used in the initial screening for filamentation was employed to determine the effects of the compounds on filamentation in serial twofold dilutions with concentrations ranging from 40 to 0.078 lM. The plates were incubated at 37 °C and examined both macroscopically and microscopically at 2, 4, and 24 h. Each assay was performed in duplicate. Microscopy Microscopy was used to directly visualize biofilms formed on the surface of the microtiter plates, as well as, the yeast or filamentous morphology of the cells in the filamentation assays. Bright-field light microscopy techniques on an inverted system microscope (Westover Scientific) equipped for photography were used.

Screening for inhibitors of C. albicans biofilms

& Caldwell, 2003) such as C. albicans biofilm formation and filamentation. This high-content screening approach requires no prior knowledge of specific molecular targets (Klebe, 1994). Instead, by screening large libraries of bioactive compounds for hits which affect a particular process of interest, it has potential for understanding the mechanisms underlying these important processes that are intimately linked to C. albicans pathogenesis. One further advantage of this approach is that it has high potential to identify new antifungal drug candidates, which are urgently needed (Pierce & Lopez-Ribot, 2013; Pierce et al., 2013). Thus, in the present study, we have performed high-content screening assays to identify inhibitors of C. albicans biofilm formation and filamentation present in different compound libraries in the Chemical Repository Collection from the National Cancer Institute. For these screenings, we used the 96-well microtiter plate model of C. albicans biofilm formation previously developed by our group, which is relatively simple and highly reproducible, and easily adaptable to high-throughput applications (Pierce et al., 2008). We also took advantage of the tight control of morphogenetic conversions in our genetically engineered C. albicans tet-NRG1 strain (Saville et al., 2003), to develop an easy, inexpensive, and robust screen for inhibitors of C. albicans filamentation, also compatible with the 96-well format.

Cytotoxicity assay Human hepatocellular carcinoma (HepG2) cells (ATCC#HB-8065) were used to determine the cytotoxicity of compounds present in the libraries. The HepG2 cells were maintained in minimum essential medium (MEM, Gibco) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate (Gibco), 19 MEM amino acid solution (Sigma), 100 IU mL 1 Penicillin, and 100 mg mL 1 streptomycin (Cellgro). The monolayers of cells were detached using 19 Trypsin/EDTA (Gibco) and centrifuged at 500 g for 7 m at 4 °C. Cell count was adjusted to 5 9 105 cells mL 1 in supplemented MEM (29) and 100 lL of the cell suspension was added to each well of white-bottomed, 96-well microtiter plates containing 100 lL of serial twofold dilutions of the small molecule compounds (100–1.56 lM) or the DMSO control. The plates were incubated for 24 h at 37 °C, and the number of viable cells was determined using the CellTiter-Glo luminescent cell viability assay (Promega). From these data, the CC50 value, defined as the concentration of each compound leading to 50% inhibition of cell viability, was calculated using GRAPHPAD PRISM software.

Results and discussion In C. albicans, genetic and genomic approaches are an important tool to study biological processes (Berman & Sudbery, 2002; Saville et al., 2005; Nobile & Mitchell, 2006; Nobile et al., 2012). However, ‘phenotype-based’ or ‘chemical genomics’ approaches in which large diverse libraries of small molecules are screened in in vitro assays developed for a particular biological process represent a valuable alternative to classical genetics, particularly for complex biological processes (Gura, 2000; Goodnow, 2001; Chanda

Initial screenings to identify inhibitors of C. albicans biofilm formation and filamentation During the initial screening, compounds from the NCI repository were identified as potential hits if they inhibited C. albicans wild-type SC5314 biofilm formation by 50% or greater in duplicate at the initial screening concentration of 5 lM. This concentration was initially chosen based on the fact that screening at medium to low concentrations normally identifies chemical matter that provides a more relevant biological (specific) starting point. This is in contrast to screening at higher concentrations, which often leads to the identification of more numerous positive signals, many of which are typically of little value (i.e. too much background noise/false positives; Goodnow, 2001). Of the 2293 compounds screened, a total of 58 initial hits were identified from the NCI libraries against C. albicans biofilm formation, resulting in a hit rate of 2.52%. Of these hits, 15 compounds were from the Natural Set (6.38% hit rate), 29 compounds from the Structural Diversity Set (1.45% hit rate), and 14 compounds from the Challenge Set (20.5% hit rate; Fig. 1). The compounds were also screened at the same initial concentration of 5 lM for inhibitors of filamentation using C. albicans tet-NRG1 strain. In this screen, control wells containing cells in the presence of doxycycline appeared cloudy due to filamentation, while cells in the absence of doxycycline remained in yeast form and fell to the bottom of the wells, forming a ring that is easily identifiable. Wells in which the compound inhibited filamentation in the cells plus doxycycline would form a ring like that of the yeast control and these compounds were identified as initial hits. A total of 41 initial hits were identified against C. albicans tet-NRG1 filamentation giving an overall hit rate of 1.79%. Eight of

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

425

Screening for inhibitors of C. albicans biofilms

(a)

C.G. Pierce et al.

hits were common inhibitors of both C. albicans biofilm formation and filamentation.

Natural Set

100% 80%

Dose–response assays for confirmation of inhibitory activity and to determine the potency and cytotoxicity of initial hits

60% 40% 20% 0%

0%

20%

(b)

40%

60%

80%

100%

80%

100%

80%

100%

Structural Diversity Set

100% 80% 60% 40% 20% 0% 0%

20%

(c)

40%

60%

Challenge Set

100% 80% 60% 40% 20% 0% 0%

20%

40%

60%

Fig. 1 Initial hits identified by screening libraries of compounds for inhibitory effects on Candida albicans biofilm formation. Candida albicans strain SC5314 biofilms were grown in the presence of compounds at a concentration of 5 lM from the (a) Natural Set, (b) Structural Diversity Set, and (c) Challenge Set. Biofilms were formed using the 96-well microtiter plate model, incubated for 24 h at 37 °C and then assessed using the XTT colorimetric assay on postwashed biofilms. Results are expressed as percent inhibition compared to control biofilms. Screening was carried out in duplicate and the results of each screen are represented on the two axes.

these compounds were from the Natural Set (3.4% hit rate), 26 from the Structural Diversity Set (1.31% hit rate), and 7 compounds were from the Challenge Set (10.29% hit rate). In these initial screenings, approximately one-third of the

426

Initial hits against C. albicans biofilm formation and filamentation were confirmed via dose–response curves. Compounds were confirmed using similar 96-well plate formats for both biofilm formation and filamentation, in which each individual hit was tested in serial twofold dilutions ranging from 0.078 to 40 lM. Of the initial 58 hits against biofilm formation, we confirmed that 24 compounds displayed a dose-dependent effect on wild-type C. albicans biofilms; and 17 of the 41 initial filamentation hits were confirmed as inhibitors using these dose–response assays. Eleven compounds inhibited both biofilm formation and filamentation in a dose-dependent manner. Thus, it is likely that some of these compounds inhibit processes other than filamentation (which is inextricably linked to biofilm formation; Lopez-Ribot, 2005) and that are also important for biofilm development, such as adhesion or matrix formation (Nett & Andes, 2006). The identity of the compounds with confirmed inhibitory activity against C. albicans biofilm formation and/ or filamentation is shown in Table 1. Figure 2 and 3 show representative microscopic observations from dose– response assays for inhibition of biofilm and filamentation, respectively, for several of the confirmed hits. Using the dose–response data generated from the XTT reduction assays on biofilms, the IC50 for each compound was determined. The IC50 values for most of the compounds were < 4.5 lM, indicating that the compounds are effective inhibitors of biofilm formation at relatively low concentrations (Table 1). Also, from the point of view of drug development, for any compound to be considered as a potential drug candidate, it must exhibit low level cytotoxicity toward human cells. Therefore, we tested toxicity of the hit compounds against a HepG2 cells. The CC50 values for each confirmed hit are also listed in Table 1. Overall, except for a few exceptions, CC50 values indicate that a majority of compounds are highly toxic even at relatively low concentrations, thus compromising their potential utility as novel therapeutic agents.

Identity of confirmed hits and their biological activity Some of the compounds from the different libraries identified during these studies deserve additional consideration, both from the point of view of their inhibitory activities on C. albicans biofilm formation and filamentation, as well as for their potential to serve as leads for antifungal drug development. We identified six confirmed hits against biofilm formation in the NCI Natural Set, and three of these hits, trichoderonin (trichodermin), nanaomycin, and rapamune (rapamycin), also inhibited filamentation in a dose–response manner. Trichodermin is an antifungal metabolite (eukaryotic protein synthesis inhibitor) produced by different fungi

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

C.G. Pierce et al.

Screening for inhibitors of C. albicans biofilms

Table 1 Confirmed inhibitors of Candida albicans biofilm formation and filamentation NSC*

Library†

Compound name/identification

Hit‡

1 2 3 4 5 6 7 8

73846 76712 267461 45737 226080 122023 4265 109509

N N N N N N SD SD

B, B B, B B, B B B,

9 10 11 12 13

35489 83217 268879 321237 293161

SD SD SD SD SD

14 15 16 17 18 19 20 21 22 23 24

18883 77552 77554 40792 40787 301460 318807 688028 323241 99843 641296

SD SD SD SD SD C C C C C C

25

693573

C

26 27

76712 677587

C C

28 29

319726 678146

C C

Trichoderonin Anisomycin Nanaomycin Bacitracin Rapamune (rapamycin, sirolimus) Valinomycin Phenanthroline Hydrochloride 17-[1-[2-(dimethylamino)ethylamino]ethyl]-13-methyl-6,7,8,9,11, 12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-ol 2-isoquinolin-2-ium-2-yl-1-phenanthren-3-ylethanone;iodide Metanilamide (3-aminobenzenesulfonamide) Mercury, (2-aminio-1,9-dihydro-6H-purine-6-thionato-N7,S6)hexylMercury, (4-amino phenyl)(6-thioguano sinato-N7,S6)2-[7-[3-(carboxymethyl)-5,10-dihydroxy-1-methyl-6,9-dioxo-3,4-dihydro-1H-benzo[g] isochromen-7-yl]-5,10-dihydroxy-1-methyl-6,9-dioxo-3,4-dihydro-1H-benzo [g]isochromen-3-yl]acetic acid 2-benzo[a]phenothiazin-12-yl-N,N-diethylethanamine 2-[ethyl(2-hydroxyethyl)amino]ethanol;2,3,4,5,6-pentachlorophenol 1,3-diaminopropan-2-ol;2,3,4,5,6-pentachlorophenol 4-amino-4-methyl-pentan-2-ol; 2,3,4,5,6-pentachlorophenol 2-methyl-N-(2-methylprop-2-enyl)prop-2-en-1-amine;2,3,4,5,6-pentachlorophenol Trichopolyn-B 1H-Azepine-1-carbothioic acid, hexa hydro-,[1-(2-pyridinyl) ethylidene]hydrazide 2-hydroxyethyl-[(2R)-2-hydroxyheptadecyl]-dimethylazanium iodide 3-Azabicyclo[3.22] nonane-3-carbo selenoic acid, [1-(2-pyridinyl)ethyidene] hydrazide Vengicide (Unamycin B, Toyocamycin) Hydrazinecarbo thioamide, N,N-dipropyl-2-(2-pyridinemethylene)-, (N,N,S)-copper(II)chloride complex, (SP-4-3)3 (4Z)-4-[[4-(dimethylamino)phenyl]methylidene]-1-methyl-2-phenylpyrazolo [1,5-a]indol-1-ium-6-ol;trifluoromethanesulfonate Anisomycin 6-Hydroxy-3-((methanesulfonyloxy) Methyl)-1-((5,6,7-tri methoxyindol-2-yl) carbonyl)indoline Azetidinecarbo thioic acid, [1-(2-pyridinyl) ethylidene] hydrazide Hexadecanaminium, 2-hydroxy-N,N-bis(2-hydroxyethyl)-N-methyl-, chloride

#

IC50 (lM)

CC50 (lM)

0.529 1.058 0.609 2.133 0.514 0.363 3.354 0.0097

1.252 0.550 0.514 286.5 67.2 2.87 1.282 8.079

B B B, F B B

3.401 3.448 0.012 1.938 3.309

13.99 106.2 3.409 4.709 96.06

B, F F F F B, B B, B, B B,

3.804 2.858 2.878 3.292 4.441 0.666 1.235 4.273 1.377 1.037 0.549

50.8 127.1 86.22 114.6 112.5 2.989 5.728 10.01 15.79 < 1.56 < 1.56

B

2.236

19.29

B B, F

0.882 0.495

B F

F F F

F

F

F F F F

2.298 ~ 9.555

0.8201 < 1.56 < 1.56 13.87

Additional information on each compound, including name, alternative names, molecular weight, molecular formula, chemical structure, chemical and physical properties, screening results in different tests, as well as available biological, pharmacological and toxicological information can be found searching by NSC number at: http://www.dtp.nci.nih.gov/dtpstandard/ChemData/index.jsp. * NCI compound identification number. † NCI libraries: Natural Set (N), Structural Diversity Set (SD), and Challenge Set (C). ‡ Confirmed hits against Biofilm Formation (B) and/or Filamentation (F).

and displays fungicide and antineoplastic activities. It has been previously reported to inhibit germ tube formation under certain environmental conditions (Shepherd et al., 1980). The antibiotic nanaomycin, produced by Streptomyces rosa var. notoensis, has previously been shown to inhibit mycoplasma, fungi, and Gram-positive bacteria growth (Tanaka et al., 1975); however, to our knowledge, its effect on C. albicans has not been described. Rapamycin is a hydrophobic macrolide antibiotic produced by the bacterium Streptomyces hygroscopicus and was first identified as an antifungal agent against C. albicans in 1975 (Vezina et al., 1975). Currently, it is used as an immunosuppressant drug. In most liquid hypha-inducing media, rapamycin has been reported to have no effect on the morphogenic transition, (Bastidas et al., 2009). Considering the fact that in the present

study, inhibitors of filamentation were identified using a nutrient rich liquid medium, the inhibition of filamentation and biofilm formation in our screening assays was most likely due to its general antifungal activity and not its role in blocking the yeast to hyphae transition. Unfortunately, the concentrations of these compounds which are toxic to the HepG2 cells, particularly trichoderonin and nanaomycin, were essentially the same as the concentrations effective at inhibiting C. albicans biofilm formation and filamentation; therefore, trichoderonin, nanaomycin (due to their toxicity), and rapamune (due to its immunosuppressive properties) would not seem viable choices as drug candidates. The compounds present in the NCI Natural set that inhibited biofilm formation only were the antibiotics anisomycin, bacitracin, and valinomycin. Anisomycin, also known as

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

427

Screening for inhibitors of C. albicans biofilms

C.G. Pierce et al.

(a)

(b)

(c)

Fig. 2 Representative microscopy pictures from dose–response experiments to confirm inhibitiors of biofilm formation. Candida albicans biofilms were grown for 24 h in the presence of a series of decreasing concentrations of the compounds and, after incubation, the wells of microtiter plates were washed and examined by light microscopy. The following compounds are depicted: (a) anisomycin, compound 2 from Natural Set; (b) metanilamide; (3-aminobenzenesulfonamide), compound 10 from Structural Diversity Set; (c) 2-benzo [a]phenothiazin-12-yl-N, N-diethylethanamine, compound 14 from Structural Diversity Set; or (d) 3-Azabicyclo [3.22] nonane-3-carbo selenoic acid, [1-(2-pyridinyl)ethyidene] hydrazide, compound 22 from Challenge Set.

(d)

flagecidin, is an antibiotic produced by Streptomyces griseolus, which inhibits protein synthesis (Grollman, 1967). Bacitracin is produced by Bacillus subtilis and is effective against Gram-positive cell walls. Although there is a wide range between the effective concentration and toxic concentration in our studies, bacitracin is known to be very toxic and cannot be administered orally. Valinomycin is a dodecapeptide antibiotic obtained from several Streptomyces strains and has been previously described to inhibit hyphal growth in C. albicans and other filamentous fungi

428

including C. tropicalis and Aureobasidium pullulans (Watanabe et al., 2005); although we did not detect an antifilamentation effect with the particular strain and conditions used in our experiments. In general, much less information is available regarding the biological activities of compounds present in the NCI’s Structural Diversity Set and Challenge Set. In the NCI Structural Diversity Set, there were seven confirmed hits against C. albicans filamentation and eight hits against biofilm formation, with three being common hits for both

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

C.G. Pierce et al.

Screening for inhibitors of C. albicans biofilms

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

Fig. 3 Representative microscopy pictures showing morphology of Candida albicans tet-NRG1 cells in the presence of 5 lM concentration of the following confirmed hit compounds: (a) trichoderonin, compound 1 from Natural Set; (b) 17-[1-[2-(dimethylamino)ethylamino] ethyl]-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-ol, compound 8 from Structural Diversity Set; (c) 2-benzo[a] phenothiazin-12-yl-N,N-diethylethanamine, compound 14 from Structural Diversity Set; (d) 2-[ethyl(2-hydroxyethyl)amino]ethanol;2,3,4,5,6-pentachlorophenol, compound 15 from Structural Diversity Set; (e) 3-Azabicyclo[3.22] nonane-3-carbo selenoic acid, [1-(2-pyridinyl)ethyidene] hydrazide, compound 22 from Challenge Set; and (f) 6-Hydroxy-3-((methanesulfonyloxy) Methyl)-1-((5,6,7-tri methoxyindol-2-yl) carbonyl)indoline, compound 27 from Challenge Set compared to control samples in the absence of compound and either (g) with no doxycycline or (h and i) plus doxycycline. Morphology of the cells in the presence of compound was examined by light microscopy after a 24 h incubation.

(Table 1). Two of these compounds were extremely potent, with IC50 values in the nanomolar range, one being an organometallic (mercury-containing) compound and the other one a compound structurally related to 17-aminoestradiols, which normally display anticoagulant and estrogenic effects (Lemini et al., 1993). Of note, we also identified a series of pentachlorophenol derivatives as inhibitors of C. albicans filamentation. Pentachlorophenol is an organochlorine compound that has been used as pesticide, fungicide, and disinfectant (Ruckdeschel & Renner, 1986). In the NCI Challenge Set, there were six hits against filamentation and 10 hits against biofilm formation with five of the hits common to both biofilm formation and filamentation (Table 1). Interestingly, one of the hits from the Challenge Set was anisomycin, a compound also identified as a hit in the Natural Set, confirming that our screening process is precise and

consistent. Two other confirmed hits from the Challenge Set were trichopolyn-B and toyocamycin. Trichopolyn-B is an antifungal peptide produced by Trichoderma polysporum with activity against C. albicans (Fuji et al., 1978; De Lucca & Walsh, 1999), and the anti-Candida activity of toyocamycin has also been previously described (Nishimura et al., 1956). Most of the compounds identified as hits also display high levels of cytotoxicity to the human cell line, even at relatively low concentrations. This was not surprising, particularly in the case of compounds from the Challenge Set, as these compounds are known to be highly toxic to different human tumor cell lines; therefore severely limiting their potential as candidates for the development of new antifungal drugs. Overall, these studies indicate that phenotypic screenings can provide chemical probes to further interrogate complex biological phenomena, such as filamentation and biofilm

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

429

Screening for inhibitors of C. albicans biofilms

formation, that are also important for the pathogenesis of C. albicans. At the same time, if toxicity issues could be overcome, some of the inhibitors identified here could represent useful leads for antifungal drug development.

Acknowledgements We thank the National Cancer Institute (NCI) for providing the compounds in the ‘NCI/DTP Open Chemical Repository’ (http://dtp.cancer.gov). We thank Amanda Tristan and Jennifer Hyde for initial assistance with screenings and thank Bruno Travi, Alex Peniche and Peter Melby for assistance with the cytotoxicity assays. This work was supported by PHS grants numbered R21DE017294 (to J.L.L.R.) and RO1AI063256 (to S.P.S.) from the National Institute of Dental & Craniofacial Research and the National Institute of Allergy and Infectious Diseases, respectively. Additional support was provided by the Army Research Office of the Department of Defense under Contract No. W911NF-11-1-0136 (to J.L.L.R. and S.P.S.) and by American Heart Association South Central Affiliate Predoctoral Fellowship number 11PRE5300004 (to C.G.P.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, and the content is solely the responsibility of the authors. References Bachmann SP, Patterson TF & Lopez-Ribot JL (2002) In vitro activity of caspofungin (MK-0991) against Candida albicans clinical isolates displaying different mechanisms of azole resistance. J Clin Microbiol 40: 2228–2230. Baillie GS & Douglas LJ (1999) Role of dimorphism in the development of Candida albicans biofilms. J Med Microbiol 48: 671–679. Bastidas RJ, Heitman J & Cardenas ME (2009) The protein kinase Tor1 regulates adhesin gene expression in Candida albicans. PLoS Pathog 5: e1000294. Berman J & Sudbery PE (2002) Candida albicans: a molecular revolution built on lessons from budding yeast. Nat Rev Genet 3: 918–930. Chanda SK & Caldwell JS (2003) Fulfilling the promise: drug discovery in the post-genomic era. Drug Discov Today 8: 168–174. De Lucca AJ & Walsh TJ (1999) Antifungal peptides: novel therapeutic compounds against emerging pathogens. Antimicrob Agents Chemother 43: 1–11. Fuji K, Fujita E, Takaishi Y, Fujita T, Arita I, Komatsu M & Hiratsuka N (1978) New antibiotics, trichopolyns A and B: isolation and biological activity. Experientia 34: 237–239. Goodnow RA Jr (2001) Current practices in generation of small molecule new leads. J Cell Biochem Suppl suppl. 37: 13–21. Grollman AP (1967) Inhibitors of protein biosynthesis. II. Mode of action of anisomycin. J Biol Chem 242: 3226–3233. Gudlaugsson O, Gillespie S, Lee K, Vande Berg J, Hu J, Messer S, Herwaldt L, Pfaller M & Diekema D (2003) Attributable mortality of nosocomial candidemia, revisited. Clin Infect Dis 37: 1172– 1177. Gura T (2000) A chemistry set for life. Nature 407: 282–284. Kadosh D & Lopez-Ribot JL (2013) Candida albicans: adapting to succeed. Cell Host Microbe 14: 483–485. Klebe G (1994) Recent developments in structure-based drug design. J Mol Med 78: 269–281.

430

C.G. Pierce et al.

Kojic EM & Darouiche RO (2004) Candida infections of medical devices. Clin Microbiol Rev 17: 255–267. Kucharikova S, Tournu H, Lagrou K, Van Dijck P & Bujdakova H (2011) Detailed comparison of Candida albicans and Candida glabrata biofilms under different conditions and their susceptibility to caspofungin and anidulafungin. J Med Microbiol 60: 1261–1269. Kucharikova S, Sharma N, Spriet I, Maertens J, Van Dijck P & Lagrou K (2013) Activities of systemically administered echinocandins against in vivo mature Candida albicans biofilms developed in a rat subcutaneous model. Antimicrob Agents Chemother 57: 2365–2368. Kuhn DM, George T, Chandra J, Mukherjee PK & Ghannoum MA (2002) Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins. Antimicrob Agents Chemother 46: 1773–1780. Lemini C, Rubio-Poo C, Silva G, Garcia-Mondragon J, Zavala E, Mendoza-Patino N, Castro D, Cruz-Almanza R & Mandoki JJ (1993) Anticoagulant and estrogenic effects of two new 17 beta-aminoestrogens, butolame [17 beta-(4-hydroxy-1-butylamino)-1,3,5(10)-estratrien-3-ol] and pentolame [17 beta-(5-hydroxy-1-pentylamino)-1,3,5 (10)-estratrien-3-ol]. Steroids 58: 457–461. Lopez-Ribot JL (2005) Candida albicans biofilms: more than filamentation. Curr Biol 15: R453–R455. Mathe L & Van Dijck P (2013) Recent insights into Candida albicans biofilm resistance mechanisms. Curr Genet 59: 251– 264. Nett J & Andes D (2006) Candida albicans biofilm development, modeling a host–pathogen interaction. Curr Opin Microbiol 9: 340–345. Nishimura H, Katagiri K, Sato K, Mayama M & Shimaoka N (1956) Toyocamycin, a new anti-candida antibiotics. J Antibiot (Tokyo) 9: 60–62. Nobile CJ & Mitchell AP (2006) Genetics and genomics of Candida albicans biofilm formation. Cell Microbiol 8: 1382–1391. Nobile CJ, Fox EP, Nett JE, Sorrells TR, Mitrovich QM, Hernday AD, Tuch BB, Andes DR & Johnson AD (2012) A recently evolved transcriptional network controls biofilm development in Candida albicans. Cell 148: 126–138. Pappas PG, Rex JH, Lee J et al. (2003) A prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients. Clin Infect Dis 37: 634–643. Pfaller MA & Diekema DJ (2007) Epidemiology of invasive candidiasis: a persistent public health problem. Clin Microbiol Rev 20: 133–163. Pierce CG & Lopez-Ribot JL (2013) Candidiasis drug discovery and development: new approaches targeting virulence for discovering and identifying new drugs. Expert Opin Drug Discov 8: 1117–1126. Pierce CG, Uppuluri P, Tristan AR, Wormley FL Jr, Mowat E, Ramage G & Lopez-Ribot JL (2008) A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing. Nat Protoc 3: 1494–1500. Pierce CG, Srinivasan A, Uppuluri P, Ramasubramanian AK & Lopez-Ribot JL (2013) Antifungal therapy with an emphasis on biofilms. Curr Opin Pharmacol 13: 726–730. Ramage G, Vande Walle K, Wickes BL & Lopez-Ribot JL (2001) Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms. Antimicrob Agents Chemother 45: 2475–2479. Ramage G, VandeWalle K, Lopez-Ribot JL & Wickes BL (2002) The filamentation pathway controlled by the Efg1 regulator protein is

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

C.G. Pierce et al.

required for normal biofilm formation and development in Candida albicans. FEMS Microbiol Lett 214: 95–100. Ramage G, Martinez JP & Lopez-Ribot JL (2006) Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res 6: 979–986. Ramage G, Mowat E, Jones B, Williams C & Lopez-Ribot J (2009) Our current understanding of fungal biofilms. Crit Rev Microbiol 35: 340–355. Ruckdeschel G & Renner G (1986) Effects of pentachlorophenol and some of its known and possible metabolites on fungi. Appl Environ Microbiol 51: 1370–1372. Saville SP, Lazzell AL, Monteagudo C & Lopez-Ribot JL (2003) Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection. Eukaryot Cell 2: 1053–1060. Saville SP, Thomas DP & Lopez-Ribot JL (2005) Use of genome information for the study of the pathogenesis of fungal infections

Screening for inhibitors of C. albicans biofilms

and the development of diagnostic tools. Rev Iberoam Micol 22: 238–241. Shepherd MG, Ghazali HM & Sullivan PA (1980) N-acetyl-D-glucosamine kinase and germ-tube formation in Candida albicans. Exp Mycol 4: 147–159. Tanaka H, Marumo H, Nagai T, Okada M, Taniguchi K & Omura S (1975) Nanaomycins, new antibiotics produced by a strain of Streptomyces. J Antibiot (Tokyo) 28: 925–930. Vezina C, Kudelski A & Sehgal SN (1975) Rapamycin (AY-22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J Antibiot (Tokyo) 28: 721–726. Watanabe H, Azuma M, Igarashi K & Ooshima H (2005) Valinomycin affects the morphology of Candida albicans. J Antibiot (Tokyo) 58: 753–758.

Pathogens and Disease (2014), 70, 423–431, © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

431

Copyright of Pathogens & Disease is the property of Wiley-Blackwell and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.

High-content phenotypic screenings to identify inhibitors of Candida albicans biofilm formation and filamentation.

Candida species represent the main cause of opportunistic fungal infections worldwide, and Candida albicans remains the most common etiological agent ...
1MB Sizes 3 Downloads 2 Views