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Understanding the cellular uptake of phosphopeptides

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Abstract

Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells—a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.

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References

  1. Davis, S. S. (1986) inDelivery Systems for Peptide Drugs (Davis, S. S., ed.), pp. 1–21. Plenum, New York.

    Google Scholar 

  2. Humphrey, M. J. (1986) inDelivery Systems for Peptide Drugs (Davis, S. S., ed.), pp. 139–151, Plenum, New York.

    Google Scholar 

  3. Kleinert, H. D., Rosenberg, S. H., Baker, W. R., Stein, H. H., Klinghofer, V., Barlow, J., et al. (1992) Discovery of a peptide-based renin inhibitor with oral bioavailability and efficacy.Science 257, 1940–1943.

    Article  PubMed  CAS  Google Scholar 

  4. Rosenberg, S. H., Kleinert, H. D., Stein, H. H., Martin, D. L., Chekal, M. A., Cohen, J., et al. (1991) Design of a well-absorbed renin inhibitor.J. Med. Chem. 34, 469–471.

    Article  PubMed  CAS  Google Scholar 

  5. Raeissi, S. and Audus, K. L. (1989) In-vitro characterization of blood-brain barrier permeability to delta sleep-inducing peptide.J. Pharm. Pharmacol. 41, 848–852.

    PubMed  CAS  Google Scholar 

  6. Cefalu, W. T. and Pardridge, W. M. (1985) Restrictive transport of a lipid-soluble peptide (cyclosporin) through the blood-brain barrier.J. Neurochem. 45(6), 1954–1956.

    Article  PubMed  CAS  Google Scholar 

  7. Ziegler, K. and Seeberger, A. (1993) Carriermediated uptake of hydrophilic linear peptides with renin inhibitory activity into isolated rat liver cells.Biochem. Pharmacol. 45(4), 909–916.

    Article  PubMed  CAS  Google Scholar 

  8. Cambell, E. B. and Griffith, O. W. (1989) Glutathione monoethyl ester: high-performance liquid chromatographic analysis and direct preparation of the free base form.Anal. Biochem. 183, 21–25.

    Article  Google Scholar 

  9. Wange, R. L., Isakov, N., Burke, T. R., Jr., Otaka, A., Roller, P. P., Watts, J. D., Aebersold, R., and Samelson, L. W. (1995) F2(Pmp)2-TAM zeta 3, a novel competitive inhibitor of the binding of ZAP-70 to the T-cell antigen receptor, blocks early T cell signaling.J. Biol. Chem. 270, 944–948.

    Article  PubMed  CAS  Google Scholar 

  10. Xiao, S., Rose, D. W., Sasaoka, T., Maegawa, H., Burke, T. R., Roller, P. P., Shoelson, S. E., and Olefsky, J. M. (1994) Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction.J. Biol. Chem. 269, 21,244–21,248.

    CAS  Google Scholar 

  11. Krise, J. P. and Stella, V. J. (1996) Prodrugs of phosphates, phosphonates, and phosphinates.Adv. Drug Deliv. Rev. 19, 287–310.

    Article  CAS  Google Scholar 

  12. Friis, G. J. and Bundgaard, H. (1996) Prodrugs pf phosphates and phosphonates: novel lipophilic α-acyloxyalkyl ester derivatives of phosphate- or phosphonate-containing drugs masking the negative charges of these groups.Eur. J. of Pharm. Sci. 4, 49–59.

    Article  CAS  Google Scholar 

  13. Kole, H. K., Akamatsu, M., Ye, B., Yan, X., Barford, D., Roller, P. P., and Burke, T. R., Jr. (1995) Protein-tyrosine phosphatase inhibition by a peptide containing the phosphotyrosyl mimetic, L-O-malonyltyrosine.Biochem. and Biophys. Res. Commun. 209(3), 817–822.

    Article  CAS  Google Scholar 

  14. Kelly, M. A., Liang, H., Sytwu, L., Vlattas, I., Lyons, N. L., Bowen, B. R., and Wennogle, L. P. (1996) Characterization of SH2-ligand interactions via library affinity selection with mass spectrometric detection.Biochem. 35(36), 11747–11755.

    Article  CAS  Google Scholar 

  15. Prochiantz, A. (1996) Getting hydrophilic compounds into cells: lessons from homeopeptides.Curr. Opinion in Neurobiol. 6, 629–634.

    Article  CAS  Google Scholar 

  16. Stewart, J. M. and Young, J. D. (1984) in Solid phase peptide synthesis, second edition, p. 103, Pierce Chemical Company, Rockford, ILL.

    Google Scholar 

  17. Bender, W. W., Garan, H., and Berg, H. C. (1971) Proteins of the human erythrocyte membrane as modified by pronase.J. Mol. Biol. 58, 783–797.

    Article  PubMed  CAS  Google Scholar 

  18. Watts, M. E., Dennis, M. F., Jones, N. R. and Stratford, M. R. (1987) A comparison of the intracellular uptake and radiosensitization efficiency in different media of uncharged 2-nitroimidizoles of varying lipophilicity.Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 52(3), 359–370.

    Article  PubMed  CAS  Google Scholar 

  19. Collis, A. J. (1993) in Medicinal Chemistry; The Role of Organic Chemistry in Drug Research (Ganellin, C. R. and Roberts, S. M., eds.), pp. 61–82, Academic Press Inc, San Diego.

    Google Scholar 

  20. Bennett, C. F. (1997) Use of cationic lipid complexes for antisense oligonucleotide delivery.Applied Antisense Oligonucleotide Technology, in press.

  21. Liu, Y., Mounkes, L. C., Liggitt, H. D., Brown, C. S., Solodin, I., Heath, T. D., and Debs, R. J. (1997) Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery.Nature Biotech. 15, 167–173.

    Article  CAS  Google Scholar 

  22. Gordon, J. A. (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor.Methods Enzymol. 201, 477–482.

    Article  PubMed  CAS  Google Scholar 

  23. Potts, R. O. and Guy, R. H. (1993) The influence of molecular volume and hydrogenbonding on peptide transport across epithelial membranes.Pharm. Res. 10(4), 635–636.

    Article  PubMed  CAS  Google Scholar 

  24. Hall, H., Williams, E. J., Moore, S. E., Walsh, F. S., Prochiantz, A., and Doherty, P. (1996) Inhibition of FGF-stimulated phosphatidylinositol hydrolysis and neurite outgrowth by a cell-membrane permeable phosphopeptide.Current Biology 6(5), 580–587.

    Article  PubMed  CAS  Google Scholar 

  25. Felgner, P. L., Holm, M., and Chan, H. (1989) Cationic liposome mediated transfection.Proc. West Pharmacol. Soc. 32, 115–121.

    PubMed  CAS  Google Scholar 

  26. Nabel, E. G., Plautz, G. E., and Nabel, G. J. (1994) Recombinant growth factor gene expression in vascular cells in vivo.Ann. NY Acad. Sci. 714, 247–252.

    Article  PubMed  CAS  Google Scholar 

  27. Caplen, N. J., Kinrade, E., Sorgi, F., Gao, X., Gruenert, D., Geddes, D., Coutelle, C., Huang, L., Alton, E. W., and Williamson, R. (1995) In vitro liposome-mediated DNA transfection of epithelial cell lines using cationic liposome DC-Chol/DOPE.Gene Ther. 2, 603–613.

    PubMed  CAS  Google Scholar 

  28. Aungst, B. J. (1993) Novel formulation strategies for improving oral bioavailability of drugs with poor membrane permeation or presystemic metabolism.J. Pharm. Sci. 82(10), 979–987.

    Article  PubMed  CAS  Google Scholar 

  29. Leick, V. (1992) Chemotactic properties, cellular binding and uptake of peptides and peptide derivatives: studies withTetrahymena thermophila.J. Cell Sci. 103, 565–570.

    PubMed  CAS  Google Scholar 

  30. Abuelyaman, A. S., Hudig, D., Woodard, S. L., and Powers, J. C. (1994) Fluorescent derivatives of diphenyl[1-(N-peptidylamino)alkyl]phosphonate esters: synthesis and use in the inhibition and cellular localization of serine proteases.Bioconjugate Chem. 5, 400–405.

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Research Department, Novartis Pharmaceuticals Division, Summit, NJ

    Alban J. Allentoff, Sreekala Mandiyan, Hongbin Liang, Anton Yuryev, Isidoros Vlattas, Timothy Duelfer, Iou-Iou Sytwu & Lawrence P. Wennogle

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  1. Alban J. Allentoff
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  2. Sreekala Mandiyan
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  3. Hongbin Liang
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  4. Anton Yuryev
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  5. Isidoros Vlattas
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  6. Timothy Duelfer
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  7. Iou-Iou Sytwu
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  8. Lawrence P. Wennogle
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Correspondence to Lawrence P. Wennogle.

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Allentoff, A.J., Mandiyan, S., Liang, H. et al. Understanding the cellular uptake of phosphopeptides. Cell Biochem Biophys 31, 129–140 (1999). https://doi.org/10.1007/BF02738168

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