Applied Microbiology and Biotechnology

, Volume 102, Issue 4, pp 1889–1901 | Cite as

Bis-guanylhydrazones as efficient anti-Candida compounds through DNA interaction

  • Jelena Lazić
  • Vladimir Ajdačić
  • Sandra Vojnovic
  • Mario Zlatović
  • Marina Pekmezovic
  • Selene Mogavero
  • Igor OpsenicaEmail author
  • Jasmina Nikodinovic-RunicEmail author
Applied microbial and cell physiology


Candida spp. are leading causes of opportunistic mycoses, including life-threatening hospital-borne infections, and novel antifungals, preferably aiming targets that have not been used before, are constantly needed. Hydrazone- and guanidine-containing molecules have shown a wide range of biological activities, including recently described excellent antifungal properties. In this study, four bis-guanylhydrazone derivatives (BG1–4) were generated following a previously developed synthetic route. Anti-Candida (two C. albicans, C. glabrata, and C. parapsilosis) minimal inhibitory concentrations (MICs) of bis-guanylhydrazones were between 2 and 15.6 μg/mL. They were also effective against preformed 48-h-old C. albicans biofilms. In vitro DNA interaction, circular dichroism, and molecular docking analysis showed the great ability of these compounds to bind fungal DNA. Competition with DNA-binding stain, exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane, and activation of metacaspases were shown for BG3. This pro-apoptotic effect of BG3 was only partially due to the accumulation of reactive oxygen species in C. albicans, as only twofold MIC and higher concentrations of BG3 caused depolarization of mitochondrial membrane which was accompanied by the decrease of the activity of fungal mitochondrial dehydrogenases, while the activity of oxidative stress response enzymes glutathione reductase and catalase was not significantly affected. BG3 showed synergistic activity with amphotericin B with a fractional inhibitory concentration index of 0.5. It also exerted low cytotoxicity and the ability to inhibit epithelial cell (TR146) invasion and damage by virulent C. albicans SC5314. With further developments, BG3 may further progress in the antifungal pipeline as a DNA-targeting agent.


Antifungal activity Candida spp. Bis-guanylhydrazone DNA interaction ROS generation Synergy 


Funding information

This work was supported by the Ministry of Education, Science and Technological Development of Serbia (Grant Nos. 172008 and 173048). Research Grant 2015 by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) to JNR is also acknowledged. Work performed in Jena was funded by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement number 642095 (OPATHY) to MP.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2018_8749_MOESM1_ESM.pdf (972 kb)
ESM 1 (PDF 972 kb)


  1. Agarwal AK, Tripathi SK, Xu T, Jacob MR, Li XC, Clark AM (2012) Exploring the molecular basis of antifungal synergies using genome-wide approaches. Front Microbiol 3:115CrossRefPubMedPubMedCentralGoogle Scholar
  2. Ajdačić V, Senerovic L, Vranić M, Pekmezovic M, Arsic-Arsenijevic V, Veselinovic A, Veselinovic J, Šolaja BA, Nikodinovic-Runic J, Opsenica IM (2016) Synthesis and evaluation of thiophene-based guanylhydrazones (iminoguanidines) efficient against panel of voriconazole-resistant fungal isolates. Bioorg Med Chem 24(6):1277–1291. CrossRefPubMedGoogle Scholar
  3. Baddley JW, Pappas PG (2005) Antifungal combination therapy: clinical potential. Drugs 65(11):1461–1480. CrossRefPubMedGoogle Scholar
  4. Bassetti M, Peghin M, Timsit JF (2016) The current treatment landscape: candidiasis. J Antimicrob Chemother 71(suppl 2):ii13–ii22CrossRefPubMedGoogle Scholar
  5. Beccia MR, Biver T, Pardini A, Spinelli J, Secco F, Venturini M, Busto Vazquez N, Lopez Cornejo MP, Martin Herrera VI, Prado Gotor R (2012) The fluorophore 4′,6-diamidino-2-phenylindole (DAPI) induces DNA folding in long double-stranded DNA. Chem Asian J 7(8):1803–1810. CrossRefPubMedGoogle Scholar
  6. Bolhuis A, Aldrich-Wright JR (2014) DNA as a target for antimicrobials. Bioorg Chem 55:51–59. CrossRefPubMedGoogle Scholar
  7. Brown GD, Denning DW, Gow NA, Levitz SM, Netea MG, White TC (2012a) Hidden killers: human fungal infections. Sci Transl Med 4(165):165rv113CrossRefGoogle Scholar
  8. Brown GD, Denning DW, Levitz SM (2012b) Tackling human fungal infections. Science 336(6082):647. CrossRefPubMedGoogle Scholar
  9. Calderone R, Sun N, Gay-Andrieu F, Groutas W, Weerawarna P, Prasad S, Alex D, Li D (2014) Antifungal drug discovery: the process and outcomes. Future Microbiol 9(6):791–805. CrossRefPubMedPubMedCentralGoogle Scholar
  10. Clinical and Laboratory Standards Institute (2008) Reference method for broth dilution antifungal susceptibility testing of yeasts—Third Edition: Approved Standard M27-A3. CLSI W, PA, USAGoogle Scholar
  11. Clinical and Laboratory Standards Institute (2012) Reference method for broth dilution antifungal susceptibility testing of yeasts: fourth informational supplement M27-S4. CLSI W, PA, USAGoogle Scholar
  12. Clinical and Laboratory Standards Institute (2015) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard—Tenth Edition M07-A10. CLSI W, PA, USAGoogle Scholar
  13. Denning DW, Bromley MJ (2015) Infectious disease. How to bolster the antifungal pipeline. Science 347(6229):1414–1416. CrossRefPubMedGoogle Scholar
  14. Denning DW, Perlin DS, Muldoon EG, Colombo AL, Chakrabarti A, Richardson MD, Sorrell TC (2017) Delivering on antimicrobial resistance agenda not possible without improving fungal diagnostic capabilities. Emerg Infect Dis 23(2):177–183. CrossRefPubMedPubMedCentralGoogle Scholar
  15. Godoy JSR, Kioshima ÉS, Abadio AKR, Felipe MSS, de Freitas SM, Svidzinski TIE (2016) Structural and functional characterization of the recombinant thioredoxin reductase from Candida albicans as a potential target for vaccine and drug design. Appl Microbiol Biotechnol 100(9):4015–4025. CrossRefPubMedGoogle Scholar
  16. Gottlieb E, Armour SM, Harris MH, Thompson CB (2003) Mitochondrial membrane potential regulates matrix configuration and cytochrome c release during apoptosis. Cell Death Differ 10(6):709–717. CrossRefPubMedGoogle Scholar
  17. Gowda KRS, Mathew BB, Sudhamani CN, Naik HSB (2014) Mechanism of DNA binding and cleavage. Biomed Biotechnol 2:1–9Google Scholar
  18. Guinea J (2014) Global trends in the distribution of Candida species causing candidemia. Clin Microbiol Infect 20:5–10. CrossRefGoogle Scholar
  19. Guo H, Xie SM, Li SX, Song YJ, Lv XL, Zhang H (2014) Synergistic mechanism for tetrandrine on fluconazole against Candida albicans through the mitochondrial aerobic respiratory metabolism pathway. J Med Microbiol 63(Pt 7):988–996. CrossRefPubMedGoogle Scholar
  20. Hansen MB, Nielsen SE, Berg K (1989) Re-examination and further development of a precise and rapid dye method for measuring cell growth/cell kill. J Immunol Methods 119(2):203–210. CrossRefPubMedGoogle Scholar
  21. Hao B, Cheng S, Clancy CJ, Nguyen MH (2013) Caspofungin kills Candida albicans by causing both cellular apoptosis and necrosis. Antimicrob Agents Chemother 57(1):326–332. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hazen KC, Lay JG, Hazen BW, RC F, Murthy S (1990) Partial biochemical characterization of cell surface hydrophobicity and hydrophilicity of Candida albicans. Infect Immun 58(11):3469–3476PubMedPubMedCentralGoogle Scholar
  23. Jakab A, Mogavero S, Forster TM, Pekmezovic M, Jablonowski N, Dombradi V, Pocsi I, Hube B (2016) Effects of the glucocorticoid betamethasone on the interaction of Candida albicans with human epithelial cells. Microbiology 162(12):2116–2125CrossRefPubMedGoogle Scholar
  24. Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta MS, Gadhwe S (2012) The biology and chemistry of antifungal agents: a review. Bioorg Med Chem 20(19):5678–5698. CrossRefPubMedGoogle Scholar
  25. Maiolo EM, Furustrand Tafin U, Borens O, Trampuz A (2014) Activities of fluconazole, caspofungin, anidulafungin, and amphotericin B on planktonic and biofilm Candida species determined by microcalorimetry. Antimicrob Agents Chemother 58(5):2709–2717. CrossRefPubMedPubMedCentralGoogle Scholar
  26. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 30(16):2785–2791. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Moyes DL, Wilson D, Richardson JP, Mogavero S, Tang SX, Wernecke J, Höfs S, Gratacap RL, Robbins J, Runglall M, Murciano C, Blagojevic M, Thavaraj S, Förster TM, Hebecker B, Kasper L, Vizcay G, Iancu SI, Kichik N, Häder A, Kurzai O, Luo T, Krüger T, Kniemeyer O, Cota E, Bader O, Wheeler RT, Gutsmann T, Hube B, Naglik JR (2016) Candidalysin is a fungal peptide toxin critical for mucosal infection. Candidalysin is a fungal peptide toxin critical for mucosal infection. Nature 532(7597):64–68. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Musiol R, Mrozek-Wilczkiewicz A, Polanski J (2014) Synergy against fungal pathogens: working together is better than working alone. Curr Med Chem 21(7):870–893. CrossRefPubMedGoogle Scholar
  29. Ngo HX, Garneau-Tsodikova S, Green KD (2016) A complex game of hide and seek: the search for new antifungals. Med Chem Commun 7(7):1285–1306. CrossRefGoogle Scholar
  30. Odds FC (2003) Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52(1):1. CrossRefPubMedGoogle Scholar
  31. Ostrosky-Zeichner L, Casadevall A, Galgiani JN, Odds FC, Rex JH (2010) An insight into the antifungal pipeline: selected new molecules and beyond. Nat Rev Drug Discov 9(9):719–727. CrossRefPubMedGoogle Scholar
  32. Perfect JR (2017) The antifungal pipeline: a reality check. Nat Rev Drug Discov 16(9):603–616. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Phillips AJ, Sudbery I, Ramsdale M (2003) Apoptosis induced by environmental stresses and amphotericin B in Candida albicans. PNAS 100(24):14327–14332. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 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(9):1494–1500. CrossRefPubMedPubMedCentralGoogle Scholar
  35. Rescifina A, Zagni C, Varrica MG, Pistarà V, Corsaro A (2014) Recent advances in small organic molecules as DNA intercalating agents: synthesis, activity, and modeling. Eur J Med Chem 74:95–115. CrossRefPubMedGoogle Scholar
  36. Rowan R, McCann M, Kavanagh K (2010) Analysis of the response of Candida albicans cells to silver(I). Med Mycol 48(3):498–505. CrossRefPubMedGoogle Scholar
  37. Sanner MF (1999) Python: a programming language for software integration and development. J Mol Graph Model 17(1):57–61PubMedGoogle Scholar
  38. Scorzoni L, de Paula ESAC, Marcos CM, Assato PA, de Melo WC, de Oliveira HC, Costa-Orlandi CB, Mendes-Giannini MJ, Fusco-Almeida AM (2017) Antifungal therapy: new advances in the understanding and treatment of mycosis. Front Microbiol 8:36CrossRefPubMedPubMedCentralGoogle Scholar
  39. Seong M, Lee DG (2018) Reactive oxygen species-independent apoptotic pathway by gold nanoparticles in Candida albicans. Microbiol Res 207(supplement C):33–40CrossRefGoogle Scholar
  40. Sharon A, Finkelstein A, Shlezinger N, Hatam I (2009) Fungal apoptosis: function, genes and gene function. FEMS Microbiol Rev 33(5):833–854. CrossRefPubMedGoogle Scholar
  41. Shirazi F, Kontoyiannis DP (2015) Micafungin triggers caspase-dependent apoptosis in Candida albicans and Candida parapsilosis biofilms, including caspofungin non-susceptible isolates. Virulence 6(4):385–394. CrossRefPubMedPubMedCentralGoogle Scholar
  42. Shrestha SK, Kril LM, Green KD, Kwiatkowski S, Sviripa VM, Nickell JR, Dwoskin LP, Watt DS, Garneau-Tsodikova S (2017) Bis(N-amidinohydrazones) and N-(amidino)-N′-aryl-bishydrazones: new classes of antibacterial/antifungal agents. Bioorg Med Chem 25(1):58–66. CrossRefPubMedGoogle Scholar
  43. Silva S, Negri M, Henriques M, Oliveira R, Williams DW, Azeredo J (2012) Candida glabrata, Candida parapsilosis and Candida tropicalis: biology, epidemiology, pathogenicity and antifungal resistance. FEMS Microbiol Rev 36(2):288–305. CrossRefPubMedGoogle Scholar
  44. Thewes S, Moran GP, Magee BB, Schaller M, Sullivan DJ, Hube B (2008) Phenotypic screening, transcriptional profiling, and comparative genomic analysis of an invasive and non-invasive strain of Candida albicans. BMC Microbiol 8(1):187. CrossRefPubMedPubMedCentralGoogle Scholar
  45. Tian H, Qu S, Wang Y, Lu Z, Zhang M, Gan Y, Zhang P, Tian J (2017) Calcium and oxidative stress mediate perillaldehyde-induced apoptosis in Candida albicans. Appl Microbiol Biotechnol 101(8):3335–3345. CrossRefPubMedGoogle Scholar
  46. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 31(2):455–461PubMedPubMedCentralGoogle Scholar
  47. Tsui C, Kong EF, Jabra-Rizk MA (2016) Pathogenesis of Candida albicans biofilm. Pathog Dis 74(4):ftw018CrossRefPubMedGoogle Scholar
  48. Uppuluri P, Srinivasan A, Ramasubramanian A, Lopez-Ribot J (2011) Effects of fluconazole, amphotericin B, and Caspofungin on Candida albicans biofilms under conditions of flow and on biofilm dispersion. Antimicrob Agents Chemother 55(7):3591–3593. CrossRefPubMedPubMedCentralGoogle Scholar
  49. van der Meer JWM, van de Veerdonk FL, Joosten LAB, Kullberg B-J, Netea MG (2010) Severe Candida spp. infections: new insights into natural immunity. Int J Antimicrob Agents 36:S58–S62. CrossRefPubMedGoogle Scholar
  50. Wachtler B, Wilson D, Haedicke K, Dalle F, Hube B (2011) From attachment to damage: defined genes of Candida albicans mediate adhesion, invasion and damage during interaction with oral epithelial cells. PLoS One 6(2):e17046. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Wachtler B, Citiulo F, Jablonowski N, Forster S, Dalle F, Schaller M, Wilson D, Hube B (2012) Candida albicans-epithelial interactions: dissecting the roles of active penetration, induced endocytosis and host factors on the infection process. PLoS One 7(5):e36952. CrossRefPubMedPubMedCentralGoogle Scholar
  52. Wei D, Wilson WD, Neidle S (2013) Small-molecule binding to the DNA minor groove is mediated by a conserved water cluster. J Am Chem Soc 135(4):1369–1377. CrossRefPubMedPubMedCentralGoogle Scholar
  53. Wu XZ, Cheng AX, Sun LM, Sun SJ, Lou HX (2009) Plagiochin E, an antifungal bis(bibenzyl), exerts its antifungal activity through mitochondrial dysfunction-induced reactive oxygen species accumulation in Candida albicans. Biochim Biophys Acta 1790:770–777CrossRefPubMedGoogle Scholar
  54. Wu XZ, Chang WQ, Cheng AX, Sun LM, Lou HX (2010) Plagiochin E, an antifungal active macrocyclic bis(bibenzyl), induced apoptosis in Candida albicans through a metacaspase-dependent apoptotic pathway. Biochim Biophys Acta 1800(4):439–347. CrossRefPubMedGoogle Scholar
  55. Wu S, Wang Y, Liu N, Dong G, Sheng C (2017) Tackling fungal resistance by biofilm inhibitors. J Med Chem 60(6):2193–2211. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Faculty of ChemistryUniversity of BelgradeBelgradeSerbia
  2. 2.Institute of Molecular Genetics and Genetic EngineeringUniversity of BelgradeBelgradeSerbia
  3. 3.Department of Microbial Pathogenicity MechanismsHans Knöll InstituteJenaGermany

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