In vitro interactions between IAP antagonist AT406 and azoles against planktonic cells and biofilms of pathogenic fungi Candida albicans and Exophiala dermatitidis

In vitro interactions between IAP antagonist AT406 and azoles against planktonic cells and... Abstract In vitro interactions of AT406, a novel IAP antagonist, and azoles including itraconazole, voriconazole, and fluconazole against planktonic cells and biofilms of Candida albicans and Exophiala dermatitidis were assessed via broth microdilution checkerboard technique. AT406 alone exhibited limited antifungal activity. However, synergistic effect between AT406 and fluconazole was observed against both planktonic cells and biofilms of C. albicans, including one fluconazole-resistant strain. Moreover, synergism was also demonstrated between AT406 and itraconazole against both planktonic cells and biofilms of E. dermatitidis. No interaction was observed between AT406 and voriconazole. No antagonism was observed in all combinations. IAP antagonist, AT406, azoles, fungi, biofilm Over the last decades, invasive fungal infection has emerged as a growing threat for human health. It's well known that fungal biofilms are relatively resistant to conventional antifungal agents.1Candida albicans has emerged as one of the major causative agents of biofilm-related infection,2 while the black yeast-like Exophiala dermatitidis is increasingly recognized as one of the biofilm-forming pathogens.3 Both the widespread use of oral triazoles and biofilm formation by opportunistic pathogenic fungus contribute to increased resistance to azole antifungal drugs and treatment failures. Nevertheless, the number of effective systemic antifungal drugs remains low. Apoptosis is a physiological process of programmed cell death critical to the normal development and maintenance of both multicellular and unicellular organisms. Inhibitors of apoptosis proteins (IAPs) characterized by the presence of Baculovirus IAP Repeat (BIR) domain, a Zn2+ ion coordinating protein–protein interaction motif, are a class of highly conserved proteins known for its important negative regulatory function in apoptosis.4 Homologues of IAPs have been identified from yeast to mammalian cells. Bir1p, the known IAP in Saccharomyces cerevisiae, has been shown to exhibit functions in yeast apoptosis, chromosome segregation, and cytokinesis.5–7 Yeast cells lacking bir1 are more sensitive to apoptosis induced by oxidative stress.5 Therefore, targeting IAPs with the goal to overcoming the evasion of apoptosis might be an attractive therapeutic strategy for developing new combinational antifungal approaches. AT406 is a novel and orally bio-available small molecular IAP antagonist, which provoke cell apoptosis by binding directly to several key IAPs to block their activities.8 In the present study, the effects of AT406 alone and combined with azoles, namely, itraconazole, voriconazole, and fluconazole, were tested against both planktonic cells and biofilms of seven isolates, including three strains of C. albicans and four strains of E. dermatitidis. C. parapsilosis ATCC 22019 was included to ensure quality control. Biofilms were prepared via a 96-well plate-based method.9 All C. albicans strains were isolated from blood samples of patients with invasive candidiasis. All E. dermatitidis isolates were also clinical isolates (three from CBS strain database and one from ATCC database). Fungal isolates were identified by microscopic morphology and by molecular sequencing of the internal transcribed spacer ribosomal DNA, as required. Broth microdilution chequerboard technique, adapted from the Clinical and Laboratory Standards Institute broth microdilution antifungal susceptibility testing was performed.10,11 Serial dilutions of AT406 (Selleck Chemicals, Houston, TX, USA), itraconazole (Selleck Chemicals), voriconazole (Selleck Chemicals), and fluconazole (Sigma Chemical Co., St. Louis, MO, USA) were prepared. The working concentration ranges against planktonic cells were 0.25–16 μg/ml for AT406, 0.03–8 μg/ml for itraconazole and voriconazole, and 0.06–32 μg/ml for fluconazole, respectively; while the working concentration ranges of AT406 and azoles against biofilms were 1–64 μg/ml and 0.125–64 μg/ml, respectively. The MICs applied for the evaluation of effects against planktonic C. albicans and E. dermatitidis were determined as the lowest concentration resulting in 50% and 100% inhibition of growth, respectively.10,11 An XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] based colorimetric assay was applied for the evaluation of effects against biofilms.12 The sessile minimum inhibitory concentration (SMIC50) was defined as the concentration at which a 50% decrease in optical density (OD) would be detected in comparison to the controls.9 The interactions between AT406 and azoles referred to the fractional inhibitory concentration index (FICI), which is classified as FICI of ≤0.5, synergy; FICI of >0.5 to ≤4, no interaction (indifference); FICI of >4, antagonism.13 In addition, synergy between azole and AT406 was also confirmed by E-test mediated susceptibility testing (AB bioMerieux, Durham, NC, USA) performed on plates with and without AT406 (4 ug/ml), as described.14 All experiments were conducted in duplicate. Table 1 shows the MICs, SMICs, and FICIs results. AT406 alone exhibited limited antifungal activity and displayed MICs of >16 μg/ml and >64 μg/ml against planktonic cells and biofilms, respectively, in both species (Table 1). However, synergism between AT406 and fluconazole was observed against all strains of C. albicans, both planktonic cells and azole-resistant biofilms. It's notable that in the fluconazole-resistant strain CA3, the presence of AT406 resulted in dramatic decrease of MIC of fluconazole from 32 μg/ml to 8 μg/ml under planktonic condition, potentiating the reversion of fluconazole resistance. As for E. dermatitidis, synergistic effects were demonstrated against both planktonic cells and azole-resistant biofilms when AT406 was combined with itraconazole. No antagonism was observed in all combinations. Table 1. Results of combinations of AT406 with azoles against Candida albicans and Exophiala dermatitidis. Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) aThe MICs for C. albicans and E. dermatitidis were defined as the concentration achieving 50% and 100% growth inhibition, respectively. bThe SMIC50 was the concentration at which a 50% decrease in absorbance would be detected in comparison to the control biofilm formed by the same fungal strain in the absence of antifungal drug. cS, synergy (FICI of ≤ 0.5); I, no interaction (indifference) (0.5 < FICI ≤ 4). For FICI calculations, the next highest concentrations of 32 μg/ml and 128 μg/ml were used as the MIC and SMIC for AT406, respectively. FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MIC, minimal inhibitory concentration; SMIC, sessile minimum inhibitory concentration; VRC, voriconazole. View Large Table 1. Results of combinations of AT406 with azoles against Candida albicans and Exophiala dermatitidis. Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) aThe MICs for C. albicans and E. dermatitidis were defined as the concentration achieving 50% and 100% growth inhibition, respectively. bThe SMIC50 was the concentration at which a 50% decrease in absorbance would be detected in comparison to the control biofilm formed by the same fungal strain in the absence of antifungal drug. cS, synergy (FICI of ≤ 0.5); I, no interaction (indifference) (0.5 < FICI ≤ 4). For FICI calculations, the next highest concentrations of 32 μg/ml and 128 μg/ml were used as the MIC and SMIC for AT406, respectively. FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MIC, minimal inhibitory concentration; SMIC, sessile minimum inhibitory concentration; VRC, voriconazole. View Large Figure 1 shows the results of E-test. As shown in Figure 1A, B, in the presence of AT406, the MIC of itraconazole against E. dermatitidis decreased from 0.38 μg/ml to 0.125 μg/ml compared to control plate. Figure 1C, D and 1E, F show the effect of the combination of AT406 and fluconazole against fluconazole-resistant C. albicans strain (CA3, MIC ≥  8 μg/ml) and fluconazole-sensitive C. albicans strain (CA2, MIC ≤ 2 μg/ml), respectively. In the fluconazole-resistant strain, the MIC of fluconazole showed dramatic decrease from 256 μg/ml to 32 μg/ml, while in the fluconazole-sensitive strain, the MIC of fluconazole also dramatically decreased from 1 μg/ml to 0.125 μg/ml in the presence of AT406. Both revealed synergism in accordance with the results of broth microdilution testings. Figure 1. View largeDownload slide E-test results of interactions between AT406 and azoles. A and B showed the MICs of itraconazole against E. dermatitidis in the absence and presence of AT406 were 0.38 μg/ml and 0.125 μg/ml, respectively. C and D showed the MICs against fluconazole-resistant C. albicans strain (CA3, MIC  ≥  8 μg/ml) in absence and presence of AT406 were 256 μg/ml and 32 μg/ml, respectively, demonstrating dramatic decrease of the MIC of fluconazole. E and F showed that in the presence of AT406, the MIC of fluconazole against fluconazole-sensitive C. albicans strain (CA2, MIC ≤ 2 μg/ml) decreased from 1 μg/ml to 0.125 μg/ml, demonstrating synergism between fluconazole and AT406. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide E-test results of interactions between AT406 and azoles. A and B showed the MICs of itraconazole against E. dermatitidis in the absence and presence of AT406 were 0.38 μg/ml and 0.125 μg/ml, respectively. C and D showed the MICs against fluconazole-resistant C. albicans strain (CA3, MIC  ≥  8 μg/ml) in absence and presence of AT406 were 256 μg/ml and 32 μg/ml, respectively, demonstrating dramatic decrease of the MIC of fluconazole. E and F showed that in the presence of AT406, the MIC of fluconazole against fluconazole-sensitive C. albicans strain (CA2, MIC ≤ 2 μg/ml) decreased from 1 μg/ml to 0.125 μg/ml, demonstrating synergism between fluconazole and AT406. This Figure is reproduced in color in the online version of Medical Mycology. AT406 was originally developed as an antitumor candidate and has been tested in Phase I clinical trial for its safety, pharmacokinetics, and pharmacodynamics in human.15 It has been demonstrated that AT406 was well tolerated at doses up to 900 mg, which achieved a Cmax of 5.6 μg/ml.15, 16 In the present study, the AT406 was tested as adjunct to conventional antifungals. Although AT406 alone showed limited antifungal activity, it did exert promising synergism with fluconazole and itraconazole against C. albicans and E. dermatitidis, respectively, both in planktonic cells and azole-resistant biofilms. Fluconazole is one of the most commonly prescribed antifungal drugs for Candida infection.17 However, fluconazole is fungistatic rather than fungicidal; therefore, treatment provides the opportunity for acquired resistance. The incidence of clinical fluconazole resistance has been estimated to be 6–36%.18,19 Thus, it's exciting to find that AT406 could result in the reversion of fluconazole resistance in planktonic cells of fluconazole-resistant C. albicans. Apoptosis has been implicated as a mechanism of posaconazole-tacrolimus or itraconazole -tacrolimus combination induced cell death in Mucorales.20 We suspected that the co-administration of pro-apoptosis IAP antagonist AT406 and inhibitors of ergosterol biosynthesis pathways might have induce more extensive apoptosis, rendering the azoles fungicidal activities. However, the underlying mechanism remains to be elucidated. In conclusion, the present study revealed that AT406 has the potential to revert fluconazole resistance in C. albicans and has a promising potential to serve as an adjunct therapy with azoles against pathogenic fungi. However, further studies are warranted to investigate the combination effects in more isolates and more species, and to evaluate the potential for concomitant use of these agents in human. Acknowledgements This work was supported by National Natural Science Foundation of China (31400131 to Lujuan Gao and 81401677 to Yi Sun), and Hubei Province Health and Family Planning Scientific Research Project (WJ2015MB281 to Yi Sun). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Davies D . Understanding biofilm resistance to antibacterial agents . Nat Rev Drug Discov . 2003 ; 2 : 114 – 122 . Google Scholar CrossRef Search ADS PubMed 2. Chen HF , Lan CY . Role of SFP1 in the Regulation of Candida albicans Biofilm Formation . PLoS One . 2015 ; 10 : e0129903 . Google Scholar CrossRef Search ADS PubMed 3. Kirchhoff L , Olsowski M , Zilmans K et al. Biofilm formation of the black yeast-like fungus Exophiala dermatitidis and its susceptibility to antiinfective agents . Sci Rep . 2017 ; 7 : 42886 . Google Scholar CrossRef Search ADS PubMed 4. Oberoi-Khanuja TK , Murali A , Rajalingam K . IAPs on the move: role of inhibitors of apoptosis proteins in cell migration . Cell Death Dis . 2013 ; 4 : e784 . Google Scholar CrossRef Search ADS PubMed 5. Walter D , Wissing S , Madeo F , Fahrenkrog B . The inhibitor-of-apoptosis protein Bir1p protects against apoptosis in S. cerevisiae and is a substrate for the yeast homologue of Omi/HtrA2 . J Cell Sci . 2006 ; 119 : 1843 – 1851 . Google Scholar CrossRef Search ADS PubMed 6. Yoon HJ , Carbon J . Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events . Proc Natl Acad Sci U S A . 1999 ; 96 : 13208 – 13213 . Google Scholar CrossRef Search ADS PubMed 7. Ren Q , Liou LC , Gao Q , Bao X , Zhang Z . Bir1 deletion causes malfunction of the spindle assembly checkpoint and apoptosis in yeast . Front Oncol . 2012 ; 2 : 93 . Google Scholar CrossRef Search ADS PubMed 8. Cai Q , Sun H , Peng Y et al. A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment . J Med Chem . 2011 ; 54 : 2714 – 2726 . Google Scholar CrossRef Search ADS PubMed 9. Pierce CG , Uppuluri P , Tristan AR et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing . Nat Protoc . 2008 ; 3 : 1494 – 1500 . Google Scholar CrossRef Search ADS PubMed 10. Clinical and Laboratory Standards Institute . Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard , 2nd ed. CLSI document M38-A2 . Wayne, PA : CLSI , 2008 . 11. Clinical and Laboratory Standards Institute . Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard , 3rd ed. CLSI document M27-A3 . Wayne, PA : CLSI , 2008 . 12. Ramage G , Vande Walle K , Wickes BL , Lopez-Ribot JL . Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms . Antimicrob Agents Chemother . 2001 ; 45 : 2475 – 2479 . Google Scholar CrossRef Search ADS PubMed 13. Odds FC . Synergy, antagonism, and what the chequerboard puts between them . J Antimicrob Chemother . 2003 ; 52 : 1 . Google Scholar CrossRef Search ADS PubMed 14. Canton E , Peman J , Gobernado M , Viudes A , Espinel-Ingroff A . Synergistic activities of fluconazole and voriconazole with terbinafine against four Candida species determined by checkerboard, time-kill, and Etest methods . Antimicrob Agents Chemother . 2005 ; 49 : 1593 – 1596 . Google Scholar CrossRef Search ADS PubMed 15. Hurwitz HI , Smith DC , Pitot HC et al. Safety, pharmacokinetics, and pharmacodynamic properties of oral DEBIO1143 (AT-406) in patients with advanced cancer: results of a first-in-man study . Cancer Chemother Pharmacol . 2015 ; 75 : 851 – 859 . Google Scholar CrossRef Search ADS PubMed 16. Jiang Y , Meng Q , Chen B et al. The small-molecule IAP antagonist AT406 inhibits pancreatic cancer cells in vitro and in vivo . Biochem Biophys Res Commun . 2016 ; 478 : 293 – 299 . Google Scholar CrossRef Search ADS PubMed 17. Pfaller MA , Diekema DJ , Gibbs DL et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion . J Clin Microbiol . 2010 ; 48 : 1366 – 1377 . Google Scholar CrossRef Search ADS PubMed 18. Baily GG , Perry FM , Denning DW , Mandal BK . Fluconazole-resistant candidosis in an HIV cohort . AIDS . 1994 ; 8 : 787 – 792 . Google Scholar CrossRef Search ADS PubMed 19. Johnson EM , Warnock DW , Luker J , Porter SR , Scully C . Emergence of azole drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis . J Antimicrob Chemother . 1995 ; 35 : 103 – 114 . Google Scholar CrossRef Search ADS PubMed 20. Shirazi F , Kontoyiannis DP . The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis . Eukaryot Cell . 2013 ; 12 : 1225 – 1234 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Medical Mycology Oxford University Press

In vitro interactions between IAP antagonist AT406 and azoles against planktonic cells and biofilms of pathogenic fungi Candida albicans and Exophiala dermatitidis

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Abstract

Abstract In vitro interactions of AT406, a novel IAP antagonist, and azoles including itraconazole, voriconazole, and fluconazole against planktonic cells and biofilms of Candida albicans and Exophiala dermatitidis were assessed via broth microdilution checkerboard technique. AT406 alone exhibited limited antifungal activity. However, synergistic effect between AT406 and fluconazole was observed against both planktonic cells and biofilms of C. albicans, including one fluconazole-resistant strain. Moreover, synergism was also demonstrated between AT406 and itraconazole against both planktonic cells and biofilms of E. dermatitidis. No interaction was observed between AT406 and voriconazole. No antagonism was observed in all combinations. IAP antagonist, AT406, azoles, fungi, biofilm Over the last decades, invasive fungal infection has emerged as a growing threat for human health. It's well known that fungal biofilms are relatively resistant to conventional antifungal agents.1Candida albicans has emerged as one of the major causative agents of biofilm-related infection,2 while the black yeast-like Exophiala dermatitidis is increasingly recognized as one of the biofilm-forming pathogens.3 Both the widespread use of oral triazoles and biofilm formation by opportunistic pathogenic fungus contribute to increased resistance to azole antifungal drugs and treatment failures. Nevertheless, the number of effective systemic antifungal drugs remains low. Apoptosis is a physiological process of programmed cell death critical to the normal development and maintenance of both multicellular and unicellular organisms. Inhibitors of apoptosis proteins (IAPs) characterized by the presence of Baculovirus IAP Repeat (BIR) domain, a Zn2+ ion coordinating protein–protein interaction motif, are a class of highly conserved proteins known for its important negative regulatory function in apoptosis.4 Homologues of IAPs have been identified from yeast to mammalian cells. Bir1p, the known IAP in Saccharomyces cerevisiae, has been shown to exhibit functions in yeast apoptosis, chromosome segregation, and cytokinesis.5–7 Yeast cells lacking bir1 are more sensitive to apoptosis induced by oxidative stress.5 Therefore, targeting IAPs with the goal to overcoming the evasion of apoptosis might be an attractive therapeutic strategy for developing new combinational antifungal approaches. AT406 is a novel and orally bio-available small molecular IAP antagonist, which provoke cell apoptosis by binding directly to several key IAPs to block their activities.8 In the present study, the effects of AT406 alone and combined with azoles, namely, itraconazole, voriconazole, and fluconazole, were tested against both planktonic cells and biofilms of seven isolates, including three strains of C. albicans and four strains of E. dermatitidis. C. parapsilosis ATCC 22019 was included to ensure quality control. Biofilms were prepared via a 96-well plate-based method.9 All C. albicans strains were isolated from blood samples of patients with invasive candidiasis. All E. dermatitidis isolates were also clinical isolates (three from CBS strain database and one from ATCC database). Fungal isolates were identified by microscopic morphology and by molecular sequencing of the internal transcribed spacer ribosomal DNA, as required. Broth microdilution chequerboard technique, adapted from the Clinical and Laboratory Standards Institute broth microdilution antifungal susceptibility testing was performed.10,11 Serial dilutions of AT406 (Selleck Chemicals, Houston, TX, USA), itraconazole (Selleck Chemicals), voriconazole (Selleck Chemicals), and fluconazole (Sigma Chemical Co., St. Louis, MO, USA) were prepared. The working concentration ranges against planktonic cells were 0.25–16 μg/ml for AT406, 0.03–8 μg/ml for itraconazole and voriconazole, and 0.06–32 μg/ml for fluconazole, respectively; while the working concentration ranges of AT406 and azoles against biofilms were 1–64 μg/ml and 0.125–64 μg/ml, respectively. The MICs applied for the evaluation of effects against planktonic C. albicans and E. dermatitidis were determined as the lowest concentration resulting in 50% and 100% inhibition of growth, respectively.10,11 An XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] based colorimetric assay was applied for the evaluation of effects against biofilms.12 The sessile minimum inhibitory concentration (SMIC50) was defined as the concentration at which a 50% decrease in optical density (OD) would be detected in comparison to the controls.9 The interactions between AT406 and azoles referred to the fractional inhibitory concentration index (FICI), which is classified as FICI of ≤0.5, synergy; FICI of >0.5 to ≤4, no interaction (indifference); FICI of >4, antagonism.13 In addition, synergy between azole and AT406 was also confirmed by E-test mediated susceptibility testing (AB bioMerieux, Durham, NC, USA) performed on plates with and without AT406 (4 ug/ml), as described.14 All experiments were conducted in duplicate. Table 1 shows the MICs, SMICs, and FICIs results. AT406 alone exhibited limited antifungal activity and displayed MICs of >16 μg/ml and >64 μg/ml against planktonic cells and biofilms, respectively, in both species (Table 1). However, synergism between AT406 and fluconazole was observed against all strains of C. albicans, both planktonic cells and azole-resistant biofilms. It's notable that in the fluconazole-resistant strain CA3, the presence of AT406 resulted in dramatic decrease of MIC of fluconazole from 32 μg/ml to 8 μg/ml under planktonic condition, potentiating the reversion of fluconazole resistance. As for E. dermatitidis, synergistic effects were demonstrated against both planktonic cells and azole-resistant biofilms when AT406 was combined with itraconazole. No antagonism was observed in all combinations. Table 1. Results of combinations of AT406 with azoles against Candida albicans and Exophiala dermatitidis. Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) aThe MICs for C. albicans and E. dermatitidis were defined as the concentration achieving 50% and 100% growth inhibition, respectively. bThe SMIC50 was the concentration at which a 50% decrease in absorbance would be detected in comparison to the control biofilm formed by the same fungal strain in the absence of antifungal drug. cS, synergy (FICI of ≤ 0.5); I, no interaction (indifference) (0.5 < FICI ≤ 4). For FICI calculations, the next highest concentrations of 32 μg/ml and 128 μg/ml were used as the MIC and SMIC for AT406, respectively. FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MIC, minimal inhibitory concentration; SMIC, sessile minimum inhibitory concentration; VRC, voriconazole. View Large Table 1. Results of combinations of AT406 with azoles against Candida albicans and Exophiala dermatitidis. Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) Strains Planktonic cells Biofilms MICa (μg/ml) MICa [A/B (μg/ml)] (FICIc) SMIC50b (μg/ml) SMIC50b [A/B (μg/ml)] (FICIc) AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC AT406 ITC VRC FLC AT406/ITC AT406/VRC AT406/FLC C. albicans  CA1 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 64/16(I) 16/16(S)  CA2 >16 0.125 0.125 0.25 2/0.125(I) 4/0.125(I) 2/0.06(S) >64 >64 >64 >64 64/16(I) 32/16(I) 16/8(S)  CA3 >16 2 0.5 32 4/2(I) 4/0.5(I) 4/8(S) >64 >64 >64 >64 32/16(I) 64/16(I) 16/16(S) E. dermatitidis  ED1 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 32/32(I) 64/64(I)  ED2 >16 1 0.5 >32 4/0.25(S) 8/0.5(I) 8/32(I) >64 >64 >64 >64 16/4(S) 32/32(I) 32/64(I)  ED3 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 8/8(S) 16/32(I) 64/64(I)  ED4 >16 1 0.5 >32 4/0.25(S) 16/0.5(I) 8/32(I) >64 >64 >64 >64 16/8(S) 16/32(I) 64/64(I) aThe MICs for C. albicans and E. dermatitidis were defined as the concentration achieving 50% and 100% growth inhibition, respectively. bThe SMIC50 was the concentration at which a 50% decrease in absorbance would be detected in comparison to the control biofilm formed by the same fungal strain in the absence of antifungal drug. cS, synergy (FICI of ≤ 0.5); I, no interaction (indifference) (0.5 < FICI ≤ 4). For FICI calculations, the next highest concentrations of 32 μg/ml and 128 μg/ml were used as the MIC and SMIC for AT406, respectively. FICI, fractional inhibitory concentration index; FLC, fluconazole; ITC, itraconazole; MIC, minimal inhibitory concentration; SMIC, sessile minimum inhibitory concentration; VRC, voriconazole. View Large Figure 1 shows the results of E-test. As shown in Figure 1A, B, in the presence of AT406, the MIC of itraconazole against E. dermatitidis decreased from 0.38 μg/ml to 0.125 μg/ml compared to control plate. Figure 1C, D and 1E, F show the effect of the combination of AT406 and fluconazole against fluconazole-resistant C. albicans strain (CA3, MIC ≥  8 μg/ml) and fluconazole-sensitive C. albicans strain (CA2, MIC ≤ 2 μg/ml), respectively. In the fluconazole-resistant strain, the MIC of fluconazole showed dramatic decrease from 256 μg/ml to 32 μg/ml, while in the fluconazole-sensitive strain, the MIC of fluconazole also dramatically decreased from 1 μg/ml to 0.125 μg/ml in the presence of AT406. Both revealed synergism in accordance with the results of broth microdilution testings. Figure 1. View largeDownload slide E-test results of interactions between AT406 and azoles. A and B showed the MICs of itraconazole against E. dermatitidis in the absence and presence of AT406 were 0.38 μg/ml and 0.125 μg/ml, respectively. C and D showed the MICs against fluconazole-resistant C. albicans strain (CA3, MIC  ≥  8 μg/ml) in absence and presence of AT406 were 256 μg/ml and 32 μg/ml, respectively, demonstrating dramatic decrease of the MIC of fluconazole. E and F showed that in the presence of AT406, the MIC of fluconazole against fluconazole-sensitive C. albicans strain (CA2, MIC ≤ 2 μg/ml) decreased from 1 μg/ml to 0.125 μg/ml, demonstrating synergism between fluconazole and AT406. This Figure is reproduced in color in the online version of Medical Mycology. Figure 1. View largeDownload slide E-test results of interactions between AT406 and azoles. A and B showed the MICs of itraconazole against E. dermatitidis in the absence and presence of AT406 were 0.38 μg/ml and 0.125 μg/ml, respectively. C and D showed the MICs against fluconazole-resistant C. albicans strain (CA3, MIC  ≥  8 μg/ml) in absence and presence of AT406 were 256 μg/ml and 32 μg/ml, respectively, demonstrating dramatic decrease of the MIC of fluconazole. E and F showed that in the presence of AT406, the MIC of fluconazole against fluconazole-sensitive C. albicans strain (CA2, MIC ≤ 2 μg/ml) decreased from 1 μg/ml to 0.125 μg/ml, demonstrating synergism between fluconazole and AT406. This Figure is reproduced in color in the online version of Medical Mycology. AT406 was originally developed as an antitumor candidate and has been tested in Phase I clinical trial for its safety, pharmacokinetics, and pharmacodynamics in human.15 It has been demonstrated that AT406 was well tolerated at doses up to 900 mg, which achieved a Cmax of 5.6 μg/ml.15, 16 In the present study, the AT406 was tested as adjunct to conventional antifungals. Although AT406 alone showed limited antifungal activity, it did exert promising synergism with fluconazole and itraconazole against C. albicans and E. dermatitidis, respectively, both in planktonic cells and azole-resistant biofilms. Fluconazole is one of the most commonly prescribed antifungal drugs for Candida infection.17 However, fluconazole is fungistatic rather than fungicidal; therefore, treatment provides the opportunity for acquired resistance. The incidence of clinical fluconazole resistance has been estimated to be 6–36%.18,19 Thus, it's exciting to find that AT406 could result in the reversion of fluconazole resistance in planktonic cells of fluconazole-resistant C. albicans. Apoptosis has been implicated as a mechanism of posaconazole-tacrolimus or itraconazole -tacrolimus combination induced cell death in Mucorales.20 We suspected that the co-administration of pro-apoptosis IAP antagonist AT406 and inhibitors of ergosterol biosynthesis pathways might have induce more extensive apoptosis, rendering the azoles fungicidal activities. However, the underlying mechanism remains to be elucidated. In conclusion, the present study revealed that AT406 has the potential to revert fluconazole resistance in C. albicans and has a promising potential to serve as an adjunct therapy with azoles against pathogenic fungi. However, further studies are warranted to investigate the combination effects in more isolates and more species, and to evaluate the potential for concomitant use of these agents in human. Acknowledgements This work was supported by National Natural Science Foundation of China (31400131 to Lujuan Gao and 81401677 to Yi Sun), and Hubei Province Health and Family Planning Scientific Research Project (WJ2015MB281 to Yi Sun). Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References 1. Davies D . Understanding biofilm resistance to antibacterial agents . Nat Rev Drug Discov . 2003 ; 2 : 114 – 122 . Google Scholar CrossRef Search ADS PubMed 2. Chen HF , Lan CY . Role of SFP1 in the Regulation of Candida albicans Biofilm Formation . PLoS One . 2015 ; 10 : e0129903 . Google Scholar CrossRef Search ADS PubMed 3. Kirchhoff L , Olsowski M , Zilmans K et al. Biofilm formation of the black yeast-like fungus Exophiala dermatitidis and its susceptibility to antiinfective agents . Sci Rep . 2017 ; 7 : 42886 . Google Scholar CrossRef Search ADS PubMed 4. Oberoi-Khanuja TK , Murali A , Rajalingam K . IAPs on the move: role of inhibitors of apoptosis proteins in cell migration . Cell Death Dis . 2013 ; 4 : e784 . Google Scholar CrossRef Search ADS PubMed 5. Walter D , Wissing S , Madeo F , Fahrenkrog B . The inhibitor-of-apoptosis protein Bir1p protects against apoptosis in S. cerevisiae and is a substrate for the yeast homologue of Omi/HtrA2 . J Cell Sci . 2006 ; 119 : 1843 – 1851 . Google Scholar CrossRef Search ADS PubMed 6. Yoon HJ , Carbon J . Participation of Bir1p, a member of the inhibitor of apoptosis family, in yeast chromosome segregation events . Proc Natl Acad Sci U S A . 1999 ; 96 : 13208 – 13213 . Google Scholar CrossRef Search ADS PubMed 7. Ren Q , Liou LC , Gao Q , Bao X , Zhang Z . Bir1 deletion causes malfunction of the spindle assembly checkpoint and apoptosis in yeast . Front Oncol . 2012 ; 2 : 93 . Google Scholar CrossRef Search ADS PubMed 8. Cai Q , Sun H , Peng Y et al. A potent and orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in clinical development for cancer treatment . J Med Chem . 2011 ; 54 : 2714 – 2726 . Google Scholar CrossRef Search ADS PubMed 9. Pierce CG , Uppuluri P , Tristan AR et al. A simple and reproducible 96-well plate-based method for the formation of fungal biofilms and its application to antifungal susceptibility testing . Nat Protoc . 2008 ; 3 : 1494 – 1500 . Google Scholar CrossRef Search ADS PubMed 10. Clinical and Laboratory Standards Institute . Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard , 2nd ed. CLSI document M38-A2 . Wayne, PA : CLSI , 2008 . 11. Clinical and Laboratory Standards Institute . Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard , 3rd ed. CLSI document M27-A3 . Wayne, PA : CLSI , 2008 . 12. Ramage G , Vande Walle K , Wickes BL , Lopez-Ribot JL . Standardized method for in vitro antifungal susceptibility testing of Candida albicans biofilms . Antimicrob Agents Chemother . 2001 ; 45 : 2475 – 2479 . Google Scholar CrossRef Search ADS PubMed 13. Odds FC . Synergy, antagonism, and what the chequerboard puts between them . J Antimicrob Chemother . 2003 ; 52 : 1 . Google Scholar CrossRef Search ADS PubMed 14. Canton E , Peman J , Gobernado M , Viudes A , Espinel-Ingroff A . Synergistic activities of fluconazole and voriconazole with terbinafine against four Candida species determined by checkerboard, time-kill, and Etest methods . Antimicrob Agents Chemother . 2005 ; 49 : 1593 – 1596 . Google Scholar CrossRef Search ADS PubMed 15. Hurwitz HI , Smith DC , Pitot HC et al. Safety, pharmacokinetics, and pharmacodynamic properties of oral DEBIO1143 (AT-406) in patients with advanced cancer: results of a first-in-man study . Cancer Chemother Pharmacol . 2015 ; 75 : 851 – 859 . Google Scholar CrossRef Search ADS PubMed 16. Jiang Y , Meng Q , Chen B et al. The small-molecule IAP antagonist AT406 inhibits pancreatic cancer cells in vitro and in vivo . Biochem Biophys Res Commun . 2016 ; 478 : 293 – 299 . Google Scholar CrossRef Search ADS PubMed 17. Pfaller MA , Diekema DJ , Gibbs DL et al. Results from the ARTEMIS DISK Global Antifungal Surveillance Study, 1997 to 2007: a 10.5-year analysis of susceptibilities of Candida species to fluconazole and voriconazole as determined by CLSI standardized disk diffusion . J Clin Microbiol . 2010 ; 48 : 1366 – 1377 . Google Scholar CrossRef Search ADS PubMed 18. Baily GG , Perry FM , Denning DW , Mandal BK . Fluconazole-resistant candidosis in an HIV cohort . AIDS . 1994 ; 8 : 787 – 792 . Google Scholar CrossRef Search ADS PubMed 19. Johnson EM , Warnock DW , Luker J , Porter SR , Scully C . Emergence of azole drug resistance in Candida species from HIV-infected patients receiving prolonged fluconazole therapy for oral candidosis . J Antimicrob Chemother . 1995 ; 35 : 103 – 114 . Google Scholar CrossRef Search ADS PubMed 20. Shirazi F , Kontoyiannis DP . The calcineurin pathway inhibitor tacrolimus enhances the in vitro activity of azoles against Mucorales via apoptosis . Eukaryot Cell . 2013 ; 12 : 1225 – 1234 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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Medical MycologyOxford University Press

Published: Jan 13, 2018

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