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Multiple Triphenylphosphonium Cations as a Platform for the Delivery of a Pro-Apoptotic Peptide

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ABSTRACT

Purpose

Triphenyl phosphonium cations (TPPs) are delocalized lipophilic cations that accumulate in the mitochondria of cells. We have explore the effect of increasing the number of TPPs on delivery of a cell-impermeable pro-apoptotic peptide to intact cells.

Methods

The pro-apoptotic peptide D-(KLAKLAK)2 (KLA) was extended with 0–3 L-Lysines modified at their ε-amine with TPP. Peptides were studied in HeLa cells to determine their cytotoxic activity and cellular uptake.

Results

In HeLa cells, the increased cytotoxicity correlates with the number of TPPs; the peptide with 3 TPP molecules (3-KLA) exerts the highest cytotoxic activity. This FITC-labeled peptide is found to accumulate in intact HeLa cells, whereas peptides with 0–2 TPPs are not detected at the same peptide concentration. Mitochondria-dependent apoptosis of HeLa cells in the presence of 3-KLA was followed by propidium iodide, Annexin-V and DiOC fluorescence by FACS.

Conclusion

A facile synthetic methodology has been presented for the delivery of a biologically active peptide into mitochondria of intact cells by attaching multiple TPP moieties to the peptide. This approach was shown to dramatically increase biological activity of the peptide as a pro-apoptotic agent.

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Abbreviations

Ahx:

6-aminohexanoicacid

Alloc:

allyloxycarbonyl

FITC:

fluorescein isothiocyanate

PI:

propidium iodide

TPP:

triphenylphosphonium

REFERENCES

  1. Ross MF, Prime TA, Abakumova I, James AM, Porteous CM, Smith RAJ, et al. Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells. Biochem J. 2008;411:633–45.

    Article  PubMed  CAS  Google Scholar 

  2. Filipovska A, Kelso GF, Brown SE, Beer SM, Smith RAJ, Murphy MP. Synthesis and characterization of a triphenylphosphonium-conjugated peroxidase mimetic. J Biol Chem. 2005;280:24113–26.

    Article  PubMed  CAS  Google Scholar 

  3. Seibel P, Trappe J, Villani G, Klopstock T, Papa S, Reichmann H. Transfection of mitochondria: strategy towards a gene therapy of mitochondrial DNA diseases. Nucl Acids Res. 1995;23:10–7.

    Article  PubMed  CAS  Google Scholar 

  4. Pathania D, Millard M, Neamati N. Opportunities in discovery and delivery of anticancer drugs targeting mitochondria and cancer cell metabolism. Adv Drug Del Rev. 2009;61:1250–75.

    Article  CAS  Google Scholar 

  5. Weissig V, Cheng S-M, D’Souza GGM. Mitochondrial pharmaceutics. Mitochondrion. 2004;3:229–44.

    Article  PubMed  CAS  Google Scholar 

  6. Mukhopadhyay A, Weiner H. Delivery of drugs and macromolecules to mitochondria. Adv Drug Del Rev. 2007;59:729–38.

    Article  CAS  Google Scholar 

  7. Hoye AT, Davoren JE, Wipf P, Fink MP, Kagan VE. Targeting Mitochondria. Acc Chem Res. 2008;41:87–97.

    Article  PubMed  CAS  Google Scholar 

  8. Torchilin VP. Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu Rev Biomed Eng. 2006;8:343–75.

    Article  PubMed  CAS  Google Scholar 

  9. Yamada Y, Akita H, Kogure K, Kamiya H, Harashima H. Mitochondrial drug delivery and mitochondrial disease therapy - an approach to liposome-based delivery targeted to mitochondria. Mitochondrion. 2007;7:63–71.

    Article  PubMed  CAS  Google Scholar 

  10. Patel NR, Hatziantoniou S, Georgopoulos A, Demetzos C, Torchilin VP, Weissig V, et al. Mitochondria-targeted liposomes improve the apoptotic and cytotoxic action of sclareol. J Liposome Res. 2010;20:244–9.

    Article  PubMed  CAS  Google Scholar 

  11. Yasuzaki Y, Yamada Y, Harashima H. Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes. Biochem Biophys Res Comm. 2010;397:181–6.

    Article  PubMed  CAS  Google Scholar 

  12. Weissig V, Boddapati SV, Cheng S-M, D’Souza GGM. Liposomes and liposome-like vesicles for drug and DNA delivery to mitochondria. J Liposome Res. 2006;16:249–64.

    Article  PubMed  CAS  Google Scholar 

  13. Yamada Y, Akita H, Kamiya H, Kogure K, Yamamoto T, Shinohara Y, et al. MITO-Porter: a liposome-based carrier system for delivery of macromolecules into mitochondria via membrane fusion. Biochim Biophys Acta. 2008;1778:423–32.

    Article  PubMed  CAS  Google Scholar 

  14. D’Souza GGM, Cheng S-M, Boddapati SV, Horobin RW, Weissig V. Nanocarrier-assisted sub-cellular targeting to the site of mitochondria improves the pro-apoptotic activity of paclitaxel. J Drug Target. 2008;16:578–85.

    Article  PubMed  Google Scholar 

  15. Hickey JL, Ruhayel RA, Barnard PJ, Baker MV, Berners-Price SJ, Filipovska A. Mitochondria-targeted chemotherapeutics: the rational design of Gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols. J Am Chem Soc. 2008;130:12570–1.

    Article  PubMed  CAS  Google Scholar 

  16. Ke H, Wang H, Wong W-K, Mak N-K, Kwong DWJ, Wong K-L, et al. Responsive and mitochondria-specific ruthenium(II) in-vitro applications: two-photon (near-infrared) induced imaging and regioselective cell killing. Chem Comm. 2010;46:6678–80.

    Article  PubMed  CAS  Google Scholar 

  17. Maiti KK, Lee WS, Takeuchi T, Watkins C, Fretz M, Kim D-C, et al. Guanidine-containing molecular transporters: sorbitol-based transporters show high intracellular selectivity toward mitochondria. Angew Chem Int Ed. 2007;46:5880–4.

    Article  CAS  Google Scholar 

  18. Sibrian-Vazquez M, Nesterova IV, Jensen TJ, Vicente MGH. Mitochondria targeting by guanidine- and biguanidine-porphyrin photosensitizers. Bioconjugate Chem. 2008;19:705–13.

    Article  CAS  Google Scholar 

  19. Fernández-Carneado J, Van Gool M, Martos V, Castel S, Prados P, de Mendoza J, et al. Highly efficient, nonpeptidic oligoguanidinium vectors that selectively internalize into mitochondria. J Am Chem Soc. 2005;127:869–74.

    Article  PubMed  Google Scholar 

  20. Biswas G, Jeon O-Y, Lee WS, Kim D-C, Kim K-T, Lee S, et al. Novel guanidine-containing molecular transporters based on lactose scaffolds: lipophilicity effect on the intracellular organellar selectivity. Chem Eur J. 2008;14:9161–8.

    Article  CAS  Google Scholar 

  21. Zhao K, Zhao G-M, Wu D, Soong Y, Birk AV, Schiller PW, et al. Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury. J Biol Chem. 2004;279:34682–90.

    Article  PubMed  CAS  Google Scholar 

  22. Szeto HH. Cell-permeable, mitochondrial-targeted, peptide antioxidants. AAPS J. 2006;8:E277–83.

    PubMed  CAS  Google Scholar 

  23. Horton KL, Stewart KM, Fonseca SB, Guo Q, Kelley SO. Mitochondria-penetrating peptides. Chem Biol. 2008;15:375–82.

    Article  PubMed  CAS  Google Scholar 

  24. Yousif LF, Stewart KM, Horton KL, Kelley SO. Mitochondria-penetrating peptides: sequence effects and model cargo transport. ChemBioChem. 2009;10:2081–8.

    Article  PubMed  CAS  Google Scholar 

  25. Yousif LF, Stewart KM, Kelley SO. Targeting mitochondria with organelle-specific compounds: strategies and applications. ChemBioChem. 2009;10:1939–50.

    Article  PubMed  CAS  Google Scholar 

  26. Horton KL, Kelley SO. Engineered apoptosis-inducing peptides with enhanced mitochondrial localization and potency. J Med Chem. 2009;52:3293–9.

    Article  PubMed  CAS  Google Scholar 

  27. Mahon KP, Potocky TB, Blair D, Roy MD, Stewart KM, Chiles TC, et al. Deconvolution of the cellular oxidative stress response with organelle-specific peptide conjugates. Chem Biol. 2007;14:923–30.

    Article  PubMed  CAS  Google Scholar 

  28. Pereira MP, Kelley SO. Maximizing the therapeutic window of an antimicrobial drug by impartig mitochondrial sequestration in human cells. J Am Chem Soc. 2011;133:3260–3.

    Article  PubMed  CAS  Google Scholar 

  29. Del Gaizo V, Payne RM. A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta. Mol Ther. 2003;7:720–30.

    Article  PubMed  Google Scholar 

  30. Mukhopadhyay A, Ni L, Yang CS, Weiner H. Bacterial signal peptide recognizes HeLa cell mitochondrial import receptors and functions as a mitochondrial leader sequence. CMLS. 2005;62:1890–9.

    Article  PubMed  CAS  Google Scholar 

  31. Flierl A, Jackson C, Cottrell B, Murdock D, Seibel P, Wallace DC. Targeted delivery of DNA to the mitochondrial compartment via import sequence-conjugated peptide nucleic acid. Mol Ther. 2003;7:550–7.

    Article  PubMed  CAS  Google Scholar 

  32. Lamla M, Seliger H, Kaufmann D. Differences in uptake, localization, and processing of PNAs modified by COX VIII pre-sequence peptide and by triphenylphoshonium cation into mitochondria of tumor cells. Drug Deliv. 2010;17:263–71.

    Article  PubMed  CAS  Google Scholar 

  33. Fetisova EK, Avetisyan AV, Izyumov DS, Korotetskaya MV, Chernyak BV, Skulachev VP. Mitochondria-targeted antioxidant SkQR1 selectively protects MDR (Pgp 170)-negative cells against oxidative stress. FEBS Lett. 2010;584:562–6.

    Article  PubMed  CAS  Google Scholar 

  34. Brown SE, Ross MF, Sanjuan-Pla A, Manas A-RB, Smith RAJ, Murphy MP. Targeting lipoic acid to mitochondria: synthesis and characterization of a triphenylphosphonium-conjugated α-lipoyl derivative. Free Rad Biol Med. 2007;42:1766–80.

    Article  PubMed  CAS  Google Scholar 

  35. Mattarei A, Biasutto L, Marotta E, De Marchi U, Sassi N, Garbisa S, et al. A mitochondriotropic derivative of Quercetin: a strategy to increase the effectiveness of polyphenols. ChemBioChem. 2008;9:2633–42.

    Article  PubMed  CAS  Google Scholar 

  36. Asin-Cayuela J, Manas A-RB, James AM, Smith RAJ, Murphy MP. Fine-tuning the hydrophobicity of a mitochondria-targeted antioxidant. FEBS Lett. 2004;571:9–16.

    Article  PubMed  CAS  Google Scholar 

  37. Murphy MP, Smith RAJ. Targeting antioxidants to mitochondria by conjugation to lipophilic cations. Annu Rev Pharmacol Toxicol. 2007;47:629–56.

    Article  PubMed  CAS  Google Scholar 

  38. Biasutto L, Mattarei A, Marotta E, Bradaschia A, Sassi N, Garbisa S, et al. Development of mitochondria-targeted derivatives of resveratrol. Bioorg Med Chem Lett. 2008;18:5594–7.

    Article  PubMed  CAS  Google Scholar 

  39. Modica-Napolitano JS, Aprille JR. Delocalized lipophilic cations selectively target the mitochondria of carcinoma cells. Adv Drug Del Rev. 2001;49:63–70.

    Article  CAS  Google Scholar 

  40. Fantin VR, Leder P. Mitochondriotoxic compounds for cancer therapy. Oncogene. 2006;25:4787–97.

    Article  PubMed  CAS  Google Scholar 

  41. Huang Z, Jiang J, Belikova N, Stoyanovsky D, Kagan V, Mintz A. Protection of normal brain cells from γ-irradiation-induced apoptosis by a mitochondria-targeted triphenyl-phosphonium-nitroxide: a possible utility in glioblastoma therapy. J Neurooncol. 2010;100:1–8.

    Article  PubMed  CAS  Google Scholar 

  42. Porteous CM, Logan A, Evans C, Ledgerwood EC, Menon DK, Aigbirhio F, et al. Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes. Biochim Biophys Acta. 2010;1800:1009–17.

    PubMed  CAS  Google Scholar 

  43. Prime TA, Blaikie FH, Evans C, Nadtochiy SM, James AM, Dahm CC, et al. A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury. Proc Natl Acad Sci USA. 2009;106:10764–9.

    Article  PubMed  CAS  Google Scholar 

  44. Belikova NA, Jiang J, Stoyanovsky DA, Glumac A, Bayir H, Greenberger JS, et al. Mitochondria-targeted (2-hydroxyamino-vinyl)-triphenyl-phosphonium releases NO and protects mouse embryonic cells against irradiation-induced apoptosis. FEBS Lett. 2009;583:1945–50.

    Article  PubMed  CAS  Google Scholar 

  45. Smith RAJ, Porteous CM, Gane AM, Murphy MP. Delivery of bioactive molecules to mitochondria in-vivo. Proc Natl Acad Sci USA. 2003;100:5407–12.

    Article  PubMed  CAS  Google Scholar 

  46. Ross MF, Filipovska A, Smith RAJ, Gait MJ, Murphy MP. Cell-penetrating peptides do not cross mitochondrial membranes even when conjugated to a lipophilic cation: evidence against direct passage through phospholipid bilayers. Biochem J. 2004;383:457–68.

    Article  PubMed  CAS  Google Scholar 

  47. Duca M, Dozza B, Lucarelli E, Santi S, Di Giorgio A, Barbarella G. Fluorescent labeling of human mesenchymal stem cells by thiophene fluorophores conjugated to a lipophilic carrier. Chem Comm. 2010;46:7948–50.

    Article  PubMed  CAS  Google Scholar 

  48. Koo C-K, So LK-Y, Wong K-L, Ho Y-M, Lam Y-W, Lam MHW, et al. A Triphenylphosphonium-functionalised cyclometalated platinum(II) complex as a nucleolus-specific two-photon molecular dye. Chem Eur J. 2010;16:3942–50.

    Article  CAS  Google Scholar 

  49. Abu-Gosh SE, Kolvazon N, Tirosh B, Ringel I, Yavin E. Multiple Triphenylphosphonium cations shuttle a hydrophilic peptide into mitochondria. Mol Pharm. 2009;6:1138–44.

    Article  PubMed  CAS  Google Scholar 

  50. Javadpour MM, Juban MM, Lo W-CJ, Bishop SM, Alberty JB, Cowell SM, et al. novo antimicrobial peptides with low mammalian cell toxicity. J Med Chem. 1996;39:3107–13.

    Article  PubMed  CAS  Google Scholar 

  51. Ellerby HM, Arap W, Ellerby LM, Kain R, Andrusiak R, Rio GD, et al. Anti-cancer activity of targeted pro-apoptotic peptides. Nature Med. 1999;5:1032–8.

    Article  PubMed  CAS  Google Scholar 

  52. Fantin VR, Berardi MJ, Babbe H, Michelman MV, Manning CM, Leder P. A bifunctional targeted peptide that blocks HER-2 tyrosine kinase and disables mitochondrial function in HER-2-positive carcinoma cells. Cancer Res. 2005;65:6891–900.

    Article  PubMed  CAS  Google Scholar 

  53. Foillard S, Jin Z-h, Garanger E, Boturyn D, Favrot M-C, Coll J-L, et al. Synthesis and biological characterisation of targeted pro-apoptotic peptide. ChemBioChem. 2008;9:2326–32.

    Article  PubMed  CAS  Google Scholar 

  54. Watkins CL, Brennan P, Fegan C, Takayama K, Nakase I, Futaki S, et al. Cellular uptake, distribution and cytotoxicity of the hydrophobic cell penetrating peptide sequence PFVYLI linked to the proapoptotic domain peptide PAD. J Controlled Rel. 2009;140:237–44.

    Article  CAS  Google Scholar 

  55. Cai H, Yang H, Xiang B, Li S, Liu S, Wan L, et al. Selective apoptotic killing of solid and hematologic tumor cells by Bombesin-targeted delivery of mitochondria-disrupting peptides. Mol Pharm. 2010;7:586–96.

    Article  PubMed  CAS  Google Scholar 

  56. Lemeshko VV. Potential-dependent membrane permeabilization and mitochondrial aggregation caused by anticancer polyarginine-KLA peptides. Arch Biochem Biophys. 2010;493:213–20.

    Article  PubMed  CAS  Google Scholar 

  57. Angeles-Boza AM, Erazo-Oliveras A, Lee Y-J, Pellois J-P. Generation of endosomolytic reagents by branching of cell-penetrating peptides: tools for the delivery of bioactive compounds to live cells in cis or trans. Bioconjugate Chem. 2010;21:2164–7.

    Article  CAS  Google Scholar 

  58. Khandazhinskaya AL, Kukhanova MK, Jasko MV. New nonnucleoside substrates for terminal deoxynucleotidyl transferase: synthesis and dependence of substrate properties on structure. Russian J Bioorg Chem. 2005;31:352–6.

    Article  CAS  Google Scholar 

  59. Zhoua P, Dragulescu-Andrasia A, Bhattacharyab B, O’Keefeb H, Vattab P, Hyldig-Nielsenb JJ, et al. Synthesis of cell-permeable peptide nucleic acids and characterization of their hybridization and uptake properties. Bioorg Med Chem Lett. 2006;16:4931–5.

    Article  Google Scholar 

  60. Jullian M, Hernandez A, Maurras A, Puget K, Amblard M, Martinez J, et al. N-terminus FITC labeling of peptides on solid support: the truth behind the spacer. Tetrahedron Lett. 2009;50:260–3.

    Article  CAS  Google Scholar 

  61. Koning AJ, Lum PY, Williams JM, Wright R. DiOC6 staining reveals organelle structure and dynamics in living yeast cells. Cell Motil Cytoskeleton. 1993;25:111–28.

    Article  PubMed  CAS  Google Scholar 

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ACKNOWLEDGMENTS

We thank the Israel Cancer Association in honor of Charles Bronfman for financial support. We thank Dr. Shai Rahimipour, Dr. Eugenia Prus and Prof. Eitan Fibach for technical assistance.

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Authors and Affiliations

  1. Institute for Drug Research, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, 91120, Israel

    Netanel Kolevzon, Uriel Kuflik, Miriam Shmuel, Sandrine Benhamron, Israel Ringel & Eylon Yavin

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  1. Netanel Kolevzon
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  2. Uriel Kuflik
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  3. Miriam Shmuel
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  4. Sandrine Benhamron
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  5. Israel Ringel
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Correspondence to Eylon Yavin.

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Kolevzon, N., Kuflik, U., Shmuel, M. et al. Multiple Triphenylphosphonium Cations as a Platform for the Delivery of a Pro-Apoptotic Peptide. Pharm Res 28, 2780–2789 (2011). https://doi.org/10.1007/s11095-011-0494-6

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