Isolation, Purification, and Characterization of Five Active Diketopiperazine Derivatives from Endophytic Streptomyces SUK 25 with Antimicrobial and Cytotoxic Activities

Muhanna M. Alshaibani; Noraziah M. Zin; Juriyati  Jalil; Nik M. Sidik; Siti Junaidah  Ahmad; Nurkhalida   Kamal  and Ruangelie  Edrada-Ebel

doi:10.4014/jmb.1608.08032

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Food Microbiology and Biotechnology(FMB) | Bioactive compounds and Physiological properties

Isolation, Purification, and Characterization of Five Active Diketopiperazine Derivatives from Endophytic Streptomyces SUK 25 with Antimicrobial and Cytotoxic Activities

Muhanna M. Alshaibani ¹, Noraziah M. Zin ^1*, Juriyati Jalil ², Nik M. Sidik ³, Siti Junaidah Ahmad ¹, Nurkhalida Kamal ⁴ and Ruangelie Edrada-Ebel ⁴

¹Programme of Biomedical Science, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia, ²Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia, ³Faculty of Ago-based Industry, Universiti Malaysia Kelantan, 17600 Jeli, Kelantan, Malaysia, ⁴Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, Scotland, ⁵Department of Pharmaceutical Sciences, Faculty of Pharmacy, Cyberjaya University College of Medical Sciences, 63000 Cyberjaya, Selangor Malaysia

Received: August 13, 2016; Accepted: May 21, 2017

J. Microbiol. Biotechnol. 2017; 27(7): 1249-1256

Published July 28, 2017 https://doi.org/10.4014/jmb.1608.08032

Abstract

In our search for new sources of bioactive secondary metabolites from Streptomyces sp., the ethyl acetate extracts from endophytic Streptomyces SUK 25 afforded five active diketopiperazine (DKP) compounds. The aim of this study was to characterize the bioactive compounds isolated from endophytic Streptomyces SUK 25 and evaluate their bioactivity against multiple drug resistance (MDR) bacteria such as Enterococcus raffinosus, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter spp., and their cytotoxic activities against the human hepatoma (HepaRG) cell line. The production of secondary metabolites by this strain was optimized through Thornton’s medium. Isolation, purification, and identification of the bioactive compounds were carried out using high-performance liquid chromatography, high-resolution mass liquid chromatography-mass spectrometry, Fourier transform infrared spectroscopy, and nuclear magnetic resonance, and cryopreserved HepaRG cells were selected to test the cytotoxicity. The results showed that endophytic Streptomyces SUK 25 produces four active DKP compounds and an acetamide derivative, which were elucidated as cyclo-(L-Val-L-Pro), cyclo- (L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Phe), and N-(7-hydroxy-6-methyl-octyl)- acetamide. These active compounds exhibited activity against methicillin-resistant S. aureus ATCC 43300 and Enterococcus raffinosus, with low toxicity against human hepatoma HepaRG cells. Endophytic Streptomyces SUK 25 has the ability to produce DKP derivatives biologically active against some MDR bacteria with relatively low toxicity against HepaRG cells line.

Keywords

Cytotoxicity, diketopiperazines, Enterococcus raffinosus, HepaRG, MRSA, Streptomyces SUK 25

References

Zin N, Loi C, Sarmin N, Rosli A. 2010. Cultivationdependent characterization of endophytic actinomycetes. Res. J. Microbiol. 5: 717-724.
Junaidah AS, Suhaini S, Sidek HM, Basri DF, Zin NM. 2015. Anti-methicillin resistant Staphylococcus aureus activity and optimal culture condition of Streptomyces sp. SUK 25. Jundishapur J. Microbiol. 8: 1-7.
Alshaibani MM, Jalil J, Sidik NM, Edrada-Ebel R, Zin NM. 2016. Isolation and characterization of cyclo-(tryptophanylprolyl) and chloramphenicol from Streptomyces sp. SUK 25 with antimethicillin-resistant Staphylococcus aureus activity. Drug Des. Devel. Ther. 10: 1817-1827.
Fenical W. 1993. Chemical studies of marine bacteria:developing a new resource. Chem. Rev. 93: 1673-1683.
Stierle A, Cardellina Ii J, Singleton F. 1988. A marine Micrococcus produces metabolites ascribed to the sponge Tedania ignis. Experientia 44: 1021-1021.
Bugni TS, Ireland CM. 2004. Marine-derived fungi: a chemically and biologically diverse group of microorganisms. Nat. Prod. Rep. 21: 143-163.
Huang R, Zhou X, Xu T, Yang X, Liu Y. 2010. Diketopiperazines from marine organisms. Chem. Biodivers. 7: 2809-2829.
Gerets H, Tilmant K, Gerin B, Chanteux H, Depelchin B, Dhalluin S, et al. 2012. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol. Toxicol. 28: 69-87.
Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C. 2007. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem. Biol. Interact. 168: 66-73.
Aninat C, Piton A, Glaise D, Le Charpentier T, Langouët S, Morel F, et al. 2006. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab. Dispos. 34: 75-83.
Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, et al. 2002. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. USA 99: 15655-15660.
Dobretsov SV, Qian P-Y. 2002. Effect of bacteria associated with the green alga Ulva reticulata on marine micro- and macrofouling. Biofouling 18: 217-228.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63.
MacIntyre L, Zhang T, Viegelmann C, Martinez IJ, Cheng C, Dowdells C, et al. 2014. Metabolomic tools for secondary metabolite discovery from marine microbial symbionts. Mar. Drugs 12: 3416-3448.
Abdelmohsen UR, Cheng C, Viegelmann C, Zhang T, Grkovic T, Ahmed S, et al. 2014. Dereplication strategies for targeted isolation of new antitrypanosomal actinosporins A and B from a marine sponge associated-Actinokineospora sp. EG49. Mar. Drugs 12: 1220-1244.
Li X, Dobretsov S, Xu Y, Xiao X, Hung OS, Qian PY. 2006. Antifouling diketopiperazines produced by a deep-sea bacterium, Streptomyces fungicidicus. Biofouling 22: 201-208.
Stark T, Hofmann T. 2005. Structures, sensory activity, and dose/response functions of 2,5-diketopiperazines in roasted cocoa nibs (Theobroma cacao). J. Agric. Food Chem. 53: 7222-7231.
Adamczeski M, Reed AR, Crews P. 1995. New and known diketopiperazines from the Caribbean sponge, Calyx cf. podatypa. J. Nat. Prod. 58: 201-208.
Rahman H. 2008. Unusual sesquiterpenes: gorgonenes and further bioactive secondary metabolites derived from marine and terrestrial bacteria. PhD Thesis. Göttingen University, Germany.
Gebhardt K, Pukall R, Fiedler H-P. 2001. Streptocidins AD, novel cyclic decapeptide antibiotics produced by Streptomyces sp. Tu 6071. I. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. (Tokyo) 54: 428-433.
Šmelcerović AA, Schiebel M, Đorđević SM. 2002. The isolation of (6S, 9S)-cyclo (prolylvalyl) from marine actinomycete, by use of high speed contercurrent chromatography. J. Serbian Chem. Soc. 67: 27-30.
Rhee K-H. 2002. Isolation and characterization of Streptomyces sp. KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics. J. Gen. Appl. Microbiol. 48: 321-327.
Jiang Z, Boyd KG, Mearns-Spragg A, Adams DR, Wright PC, Burgess JG. 2000. Two diketopiperazines and one halogenated phenol from cultures of the marine bacterium, Pseudoalteromonas luteoviolacea. Nat. Prod. Lett. 14: 435-440.
Rhee K-H. 2004. Cyclic dipeptides exhibit synergistic, broad spectrum antimicrobial effects and have anti-mutagenic properties. Int. J. Antimicrob. Agents 24: 423-427.
Furtado NA, Pupo MT, Carvalho I, Campo VL, Duarte MCT, Bastos JK. 2005. Diketopiperazines produced by an Aspergillus fumigatus Brazilian strain. J. Braz. Chem. Soc. 16: 1448-1453.
de Carvalho MP, Abraham W-R. 2012. Antimicrobial and biofilm inhibiting diketopiperazines. Curr. Med. Chem. 19: 3564-3577.
Huang R, Yan T, Peng Y, Zhou X, Yang X, Liu Y. 2014. Diketopiperazines from the marine sponge Axinella sp. Chem. Nat. Compd. 50: 191-193.
Khedr AI, Mohamed GA, Orabi MA, Ibrahim SR, Yamada K. 2015. Staphylopeptide A, a new cyclic tetrapeptide from culture broth of Staphylococcus sp. Phytochem. Lett. 13: 11-14.
Szabo M, Veres Z, Baranyai Z, Jakab F, Jemnitz K. 2013. Comparison of human hepatoma HepaRG cells with human and rat hepatocytes in uptake transport assays in order to predict a risk of drug induced hepatotoxicity. PLoS One 8: e59432.
Vázquez-Rivera D, González O, Guzmán-Rodríguez J, DíazPérez AL, Ochoa-Zarzosa A, López-Bucio J, et al. 2015. Cytotoxicity of cyclodipeptides from Pseudomonas aeruginosa PAO1 leads to apoptosis in human cancer cell lines. Biomed. Res. Int. 2015: 197608.
Cui CB, Usukata M, Kakeya H, Onose R, Okada G, Takahashi I, et al. 1996. Acetophthalidin, a novel inhibitor of mammalian cell cycle, produced by a fungus isolated from a sea sediment. J. Antibiot. (Tokyo) 49: 216-219.
Kondoh M, Usui T, Mayumi T, Osada H. 1998. Effects of tryprostatin derivatives on microtubule assembly in vitro and in situ. J. Antibiot. (Tokyo) 51: 801-804.
Folkes A, Brown SD, Canne LE, Chan J, Engelhardt E, Epshteyn S, et al. 2002. Design, synthesis and in vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1. Bioorg. Med. Chem. Lett. 12: 1063-1066.
Martins MB, Carvalho I. 2007. Diketopiperazines: biological activity and synthesis. Tetrahedron 63: 9923-9932.
McCleland K, Milne P, Lucieto F, Frost C, Brauns S, Venter M, et al. 2004. An investigation into the biological activity of the selected histidine-containing diketopiperazines cyclo (HisPhe) and cyclo (His-Tyr). J. Pharm. Pharmacol. 56: 1143-1153.
Choi E, Park JS, Kim YJ, Jung JH, Lee J, Kwon H, et al. 2011. Apoptosis-inducing effect of diketopiperazine disulfides produced by Aspergillus sp. KMD 901 isolated from marine sediment on HCT116 colon cancer cell lines. J. Appl. Microbiol. 110: 304-313.

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Research article

J. Microbiol. Biotechnol. 2017; 27(7): 1249-1256

Published online July 28, 2017 https://doi.org/10.4014/jmb.1608.08032

Isolation, Purification, and Characterization of Five Active Diketopiperazine Derivatives from Endophytic Streptomyces SUK 25 with Antimicrobial and Cytotoxic Activities

Muhanna M. Alshaibani ¹, Noraziah M. Zin ^1*, Juriyati Jalil ², Nik M. Sidik ³, Siti Junaidah Ahmad ¹, Nurkhalida Kamal ⁴ and Ruangelie Edrada-Ebel ⁴

¹Programme of Biomedical Science, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia, ²Drug and Herbal Research Centre, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia, ³Faculty of Ago-based Industry, Universiti Malaysia Kelantan, 17600 Jeli, Kelantan, Malaysia, ⁴Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow G4 0RE, Scotland, ⁵Department of Pharmaceutical Sciences, Faculty of Pharmacy, Cyberjaya University College of Medical Sciences, 63000 Cyberjaya, Selangor Malaysia

Received: August 13, 2016; Accepted: May 21, 2017

Abstract

In our search for new sources of bioactive secondary metabolites from Streptomyces sp., the
ethyl acetate extracts from endophytic Streptomyces SUK 25 afforded five active
diketopiperazine (DKP) compounds. The aim of this study was to characterize the bioactive
compounds isolated from endophytic Streptomyces SUK 25 and evaluate their bioactivity
against multiple drug resistance (MDR) bacteria such as Enterococcus raffinosus, Staphylococcus
aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter
spp., and their cytotoxic activities against the human hepatoma (HepaRG) cell line. The
production of secondary metabolites by this strain was optimized through Thornton’s
medium. Isolation, purification, and identification of the bioactive compounds were carried
out using high-performance liquid chromatography, high-resolution mass liquid
chromatography-mass spectrometry, Fourier transform infrared spectroscopy, and nuclear
magnetic resonance, and cryopreserved HepaRG cells were selected to test the cytotoxicity.
The results showed that endophytic Streptomyces SUK 25 produces four active DKP
compounds and an acetamide derivative, which were elucidated as cyclo-(L-Val-L-Pro), cyclo-
(L-Leu-L-Pro), cyclo-(L-Phe-L-Pro), cyclo-(L-Val-L-Phe), and N-(7-hydroxy-6-methyl-octyl)-
acetamide. These active compounds exhibited activity against methicillin-resistant S. aureus
ATCC 43300 and Enterococcus raffinosus, with low toxicity against human hepatoma HepaRG
cells. Endophytic Streptomyces SUK 25 has the ability to produce DKP derivatives biologically
active against some MDR bacteria with relatively low toxicity against HepaRG cells line.

Keywords: Cytotoxicity, diketopiperazines, Enterococcus raffinosus, HepaRG, MRSA, Streptomyces SUK 25

Abstract

References

Zin N, Loi C, Sarmin N, Rosli A. 2010. Cultivationdependent characterization of endophytic actinomycetes. Res. J. Microbiol. 5: 717-724.
Junaidah AS, Suhaini S, Sidek HM, Basri DF, Zin NM. 2015. Anti-methicillin resistant Staphylococcus aureus activity and optimal culture condition of Streptomyces sp. SUK 25. Jundishapur J. Microbiol. 8: 1-7.
Alshaibani MM, Jalil J, Sidik NM, Edrada-Ebel R, Zin NM. 2016. Isolation and characterization of cyclo-(tryptophanylprolyl) and chloramphenicol from Streptomyces sp. SUK 25 with antimethicillin-resistant Staphylococcus aureus activity. Drug Des. Devel. Ther. 10: 1817-1827.
Fenical W. 1993. Chemical studies of marine bacteria:developing a new resource. Chem. Rev. 93: 1673-1683.
Stierle A, Cardellina Ii J, Singleton F. 1988. A marine Micrococcus produces metabolites ascribed to the sponge Tedania ignis. Experientia 44: 1021-1021.
Bugni TS, Ireland CM. 2004. Marine-derived fungi: a chemically and biologically diverse group of microorganisms. Nat. Prod. Rep. 21: 143-163.
Huang R, Zhou X, Xu T, Yang X, Liu Y. 2010. Diketopiperazines from marine organisms. Chem. Biodivers. 7: 2809-2829.
Gerets H, Tilmant K, Gerin B, Chanteux H, Depelchin B, Dhalluin S, et al. 2012. Characterization of primary human hepatocytes, HepG2 cells, and HepaRG cells at the mRNA level and CYP activity in response to inducers and their predictivity for the detection of human hepatotoxins. Cell Biol. Toxicol. 28: 69-87.
Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C. 2007. The human hepatoma HepaRG cells: a highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chem. Biol. Interact. 168: 66-73.
Aninat C, Piton A, Glaise D, Le Charpentier T, Langouët S, Morel F, et al. 2006. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab. Dispos. 34: 75-83.
Gripon P, Rumin S, Urban S, Le Seyec J, Glaise D, Cannie I, et al. 2002. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. USA 99: 15655-15660.
Dobretsov SV, Qian P-Y. 2002. Effect of bacteria associated with the green alga Ulva reticulata on marine micro- and macrofouling. Biofouling 18: 217-228.
Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 65: 55-63.
MacIntyre L, Zhang T, Viegelmann C, Martinez IJ, Cheng C, Dowdells C, et al. 2014. Metabolomic tools for secondary metabolite discovery from marine microbial symbionts. Mar. Drugs 12: 3416-3448.
Abdelmohsen UR, Cheng C, Viegelmann C, Zhang T, Grkovic T, Ahmed S, et al. 2014. Dereplication strategies for targeted isolation of new antitrypanosomal actinosporins A and B from a marine sponge associated-Actinokineospora sp. EG49. Mar. Drugs 12: 1220-1244.
Li X, Dobretsov S, Xu Y, Xiao X, Hung OS, Qian PY. 2006. Antifouling diketopiperazines produced by a deep-sea bacterium, Streptomyces fungicidicus. Biofouling 22: 201-208.
Stark T, Hofmann T. 2005. Structures, sensory activity, and dose/response functions of 2,5-diketopiperazines in roasted cocoa nibs (Theobroma cacao). J. Agric. Food Chem. 53: 7222-7231.
Adamczeski M, Reed AR, Crews P. 1995. New and known diketopiperazines from the Caribbean sponge, Calyx cf. podatypa. J. Nat. Prod. 58: 201-208.
Rahman H. 2008. Unusual sesquiterpenes: gorgonenes and further bioactive secondary metabolites derived from marine and terrestrial bacteria. PhD Thesis. Göttingen University, Germany.
Gebhardt K, Pukall R, Fiedler H-P. 2001. Streptocidins AD, novel cyclic decapeptide antibiotics produced by Streptomyces sp. Tu 6071. I. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. (Tokyo) 54: 428-433.
Šmelcerović AA, Schiebel M, Đorđević SM. 2002. The isolation of (6S, 9S)-cyclo (prolylvalyl) from marine actinomycete, by use of high speed contercurrent chromatography. J. Serbian Chem. Soc. 67: 27-30.
Rhee K-H. 2002. Isolation and characterization of Streptomyces sp. KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics. J. Gen. Appl. Microbiol. 48: 321-327.
Jiang Z, Boyd KG, Mearns-Spragg A, Adams DR, Wright PC, Burgess JG. 2000. Two diketopiperazines and one halogenated phenol from cultures of the marine bacterium, Pseudoalteromonas luteoviolacea. Nat. Prod. Lett. 14: 435-440.
Rhee K-H. 2004. Cyclic dipeptides exhibit synergistic, broad spectrum antimicrobial effects and have anti-mutagenic properties. Int. J. Antimicrob. Agents 24: 423-427.
Furtado NA, Pupo MT, Carvalho I, Campo VL, Duarte MCT, Bastos JK. 2005. Diketopiperazines produced by an Aspergillus fumigatus Brazilian strain. J. Braz. Chem. Soc. 16: 1448-1453.
de Carvalho MP, Abraham W-R. 2012. Antimicrobial and biofilm inhibiting diketopiperazines. Curr. Med. Chem. 19: 3564-3577.
Huang R, Yan T, Peng Y, Zhou X, Yang X, Liu Y. 2014. Diketopiperazines from the marine sponge Axinella sp. Chem. Nat. Compd. 50: 191-193.
Khedr AI, Mohamed GA, Orabi MA, Ibrahim SR, Yamada K. 2015. Staphylopeptide A, a new cyclic tetrapeptide from culture broth of Staphylococcus sp. Phytochem. Lett. 13: 11-14.
Szabo M, Veres Z, Baranyai Z, Jakab F, Jemnitz K. 2013. Comparison of human hepatoma HepaRG cells with human and rat hepatocytes in uptake transport assays in order to predict a risk of drug induced hepatotoxicity. PLoS One 8: e59432.
Vázquez-Rivera D, González O, Guzmán-Rodríguez J, DíazPérez AL, Ochoa-Zarzosa A, López-Bucio J, et al. 2015. Cytotoxicity of cyclodipeptides from Pseudomonas aeruginosa PAO1 leads to apoptosis in human cancer cell lines. Biomed. Res. Int. 2015: 197608.
Cui CB, Usukata M, Kakeya H, Onose R, Okada G, Takahashi I, et al. 1996. Acetophthalidin, a novel inhibitor of mammalian cell cycle, produced by a fungus isolated from a sea sediment. J. Antibiot. (Tokyo) 49: 216-219.
Kondoh M, Usui T, Mayumi T, Osada H. 1998. Effects of tryprostatin derivatives on microtubule assembly in vitro and in situ. J. Antibiot. (Tokyo) 51: 801-804.
Folkes A, Brown SD, Canne LE, Chan J, Engelhardt E, Epshteyn S, et al. 2002. Design, synthesis and in vitro evaluation of potent, novel, small molecule inhibitors of plasminogen activator inhibitor-1. Bioorg. Med. Chem. Lett. 12: 1063-1066.
Martins MB, Carvalho I. 2007. Diketopiperazines: biological activity and synthesis. Tetrahedron 63: 9923-9932.
McCleland K, Milne P, Lucieto F, Frost C, Brauns S, Venter M, et al. 2004. An investigation into the biological activity of the selected histidine-containing diketopiperazines cyclo (HisPhe) and cyclo (His-Tyr). J. Pharm. Pharmacol. 56: 1143-1153.
Choi E, Park JS, Kim YJ, Jung JH, Lee J, Kwon H, et al. 2011. Apoptosis-inducing effect of diketopiperazine disulfides produced by Aspergillus sp. KMD 901 isolated from marine sediment on HCT116 colon cancer cell lines. J. Appl. Microbiol. 110: 304-313.