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. 1998 Aug 1;333(Pt 3):621–630. doi: 10.1042/bj3330621

Linear and cyclic peptides as substrates and modulators of P-glycoprotein: peptide binding and effects on drug transport and accumulation.

F J Sharom 1, P Lu 1, R Liu 1, X Yu 1
PMCID: PMC1219625  PMID: 9677321

Abstract

One cause of multidrug resistance (MDR) in human cancers is the overexpression of the P-glycoprotein multidrug transporter, a member of the ABC superfamily of membrane proteins. Natural products and chemotherapeutic drugs are pumped out of the cell by P-glycoprotein in an ATP-dependent fashion. There is growing evidence that many hydrophobic peptides are also P-glycoprotein substrates. With the use of a fluorescence-quenching assay, we have shown that some linear and cyclic hydrophobic peptides interact with P-glycoprotein, whereas others do not. The measured values of the quenching constant, Kq, for interaction of peptides with P-glycoprotein ranged from 200 nM for cyclosporine A to 138 microM for the tripeptide N-acetyl-leucyl-leucyl-norleucinal. Peptides that interacted with P-glycoprotein in the fluorescence assay also blocked colchicine transport into plasma membrane vesicles from MDR cells. The values of Dm, the peptide concentration causing 50% inhibition of drug uptake, were highly correlated with the values of Kq, over three orders of magnitude. The P-glycoprotein ATPase stimulation/inhibition profile of the peptides was not helpful in making a quantitative assessment of the ability of a peptide to interact with P-glycoprotein or to block drug transport. Some hydrophobic peptides were able to restore accumulation in MDR cells of the chemotherapeutic drug daunorubicin and the fluorescent dye rhodamine 123 to the levels observed in the drug-sensitive parent. Peptides that interacted with P-glycoprotein also displayed a relatively low overall toxicity to intact MDR cells, and inhibited drug transport at concentrations below the toxic range. Hydrophobic peptides should be given serious consideration for development as clinical chemosensitizing agents.

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Selected References

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  1. Boer R., Ulrich W. R., Haas S., Borchers C., Gekeler V., Boss H., Przybylski M., Schödl A. Interaction of cytostatics and chemosensitizers with the dexniguldipine binding site on P-glycoprotein. Eur J Pharmacol. 1996 Jan 11;295(2-3):253–260. doi: 10.1016/0014-2999(95)00649-4. [DOI] [PubMed] [Google Scholar]
  2. Bosch I., Croop J. P-glycoprotein multidrug resistance and cancer. Biochim Biophys Acta. 1996 Oct 9;1288(2):F37–F54. doi: 10.1016/0304-419x(96)00022-4. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Chou T. C. Derivation and properties of Michaelis-Menten type and Hill type equations for reference ligands. J Theor Biol. 1976 Jul 7;59(2):253–276. doi: 10.1016/0022-5193(76)90169-7. [DOI] [PubMed] [Google Scholar]
  5. Dhir R., Grizzuti K., Kajiji S., Gros P. Modulatory effects on substrate specificity of independent mutations at the serine939/941 position in predicted transmembrane domain 11 of P-glycoproteins. Biochemistry. 1993 Sep 14;32(36):9492–9499. doi: 10.1021/bi00087a030. [DOI] [PubMed] [Google Scholar]
  6. DiDiodato G., Sharom F. J. Interaction of combinations of drugs, chemosensitizers, and peptides with the P-glycoprotein multidrug transporter. Biochem Pharmacol. 1997 Jun 15;53(12):1789–1797. doi: 10.1016/s0006-2952(97)00007-5. [DOI] [PubMed] [Google Scholar]
  7. Doige C. A., Ames G. F. ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance. Annu Rev Microbiol. 1993;47:291–319. doi: 10.1146/annurev.mi.47.100193.001451. [DOI] [PubMed] [Google Scholar]
  8. Doige C. A., Sharom F. J. Strategies for the purification of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. Protein Expr Purif. 1991 Aug;2(4):256–265. doi: 10.1016/1046-5928(91)90081-s. [DOI] [PubMed] [Google Scholar]
  9. Doige C. A., Yu X., Sharom F. J. ATPase activity of partially purified P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. Biochim Biophys Acta. 1992 Aug 24;1109(2):149–160. doi: 10.1016/0005-2736(92)90078-z. [DOI] [PubMed] [Google Scholar]
  10. Eytan G. D., Borgnia M. J., Regev R., Assaraf Y. G. Transport of polypeptide ionophores into proteoliposomes reconstituted with rat liver P-glycoprotein. J Biol Chem. 1994 Oct 21;269(42):26058–26065. [PubMed] [Google Scholar]
  11. Eytan G. D., Regev R., Oren G., Assaraf Y. G. The role of passive transbilayer drug movement in multidrug resistance and its modulation. J Biol Chem. 1996 May 31;271(22):12897–12902. doi: 10.1074/jbc.271.22.12897. [DOI] [PubMed] [Google Scholar]
  12. Ford J. M. Experimental reversal of P-glycoprotein-mediated multidrug resistance by pharmacological chemosensitisers. Eur J Cancer. 1996 Jun;32A(6):991–1001. doi: 10.1016/0959-8049(96)00047-0. [DOI] [PubMed] [Google Scholar]
  13. Germann U. A. P-glycoprotein--a mediator of multidrug resistance in tumour cells. Eur J Cancer. 1996 Jun;32A(6):927–944. doi: 10.1016/0959-8049(96)00057-3. [DOI] [PubMed] [Google Scholar]
  14. Higgins C. F., Gottesman M. M. Is the multidrug transporter a flippase? Trends Biochem Sci. 1992 Jan;17(1):18–21. doi: 10.1016/0968-0004(92)90419-a. [DOI] [PubMed] [Google Scholar]
  15. Homolya L., Holló Z., Germann U. A., Pastan I., Gottesman M. M., Sarkadi B. Fluorescent cellular indicators are extruded by the multidrug resistance protein. J Biol Chem. 1993 Oct 15;268(29):21493–21496. [PubMed] [Google Scholar]
  16. Kartner N., Evernden-Porelle D., Bradley G., Ling V. Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies. 1985 Aug 29-Sep 4Nature. 316(6031):820–823. doi: 10.1038/316820a0. [DOI] [PubMed] [Google Scholar]
  17. Leveille-Webster C. R., Arias I. M. The biology of the P-glycoproteins. J Membr Biol. 1995 Jan;143(2):89–102. doi: 10.1007/BF00234655. [DOI] [PubMed] [Google Scholar]
  18. Ling V., Thompson L. H. Reduced permeability in CHO cells as a mechanism of resistance to colchicine. J Cell Physiol. 1974 Feb;83(1):103–116. doi: 10.1002/jcp.1040830114. [DOI] [PubMed] [Google Scholar]
  19. Liu R., Sharom F. J. Fluorescence studies on the nucleotide binding domains of the P-glycoprotein multidrug transporter. Biochemistry. 1997 Mar 11;36(10):2836–2843. doi: 10.1021/bi9627119. [DOI] [PubMed] [Google Scholar]
  20. Liu R., Sharom F. J. Site-directed fluorescence labeling of P-glycoprotein on cysteine residues in the nucleotide binding domains. Biochemistry. 1996 Sep 10;35(36):11865–11873. doi: 10.1021/bi960823u. [DOI] [PubMed] [Google Scholar]
  21. Loe D. W., Sharom F. J. Interaction of multidrug-resistant Chinese hamster ovary cells with amphiphiles. Br J Cancer. 1993 Aug;68(2):342–351. doi: 10.1038/bjc.1993.338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Loe D. W., Sharom F. J. Interaction of multidrug-resistant Chinese hamster ovary cells with the peptide ionophore gramicidin D. Biochim Biophys Acta. 1994 Feb 23;1190(1):72–84. doi: 10.1016/0005-2736(94)90035-3. [DOI] [PubMed] [Google Scholar]
  23. Loo T. W., Clarke D. M. Functional consequences of phenylalanine mutations in the predicted transmembrane domain of P-glycoprotein. J Biol Chem. 1993 Sep 25;268(27):19965–19972. [PubMed] [Google Scholar]
  24. Loo T. W., Clarke D. M. Functional consequences of proline mutations in the predicted transmembrane domain of P-glycoprotein. J Biol Chem. 1993 Feb 15;268(5):3143–3149. [PubMed] [Google Scholar]
  25. Loo T. W., Clarke D. M. Reconstitution of drug-stimulated ATPase activity following co-expression of each half of human P-glycoprotein as separate polypeptides. J Biol Chem. 1994 Mar 11;269(10):7750–7755. [PubMed] [Google Scholar]
  26. Pearce H. L., Safa A. R., Bach N. J., Winter M. A., Cirtain M. C., Beck W. T. Essential features of the P-glycoprotein pharmacophore as defined by a series of reserpine analogs that modulate multidrug resistance. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5128–5132. doi: 10.1073/pnas.86.13.5128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Peterson G. L. Determination of total protein. Methods Enzymol. 1983;91:95–119. doi: 10.1016/s0076-6879(83)91014-5. [DOI] [PubMed] [Google Scholar]
  28. Raviv Y., Pollard H. B., Bruggemann E. P., Pastan I., Gottesman M. M. Photosensitized labeling of a functional multidrug transporter in living drug-resistant tumor cells. J Biol Chem. 1990 Mar 5;265(7):3975–3980. [PubMed] [Google Scholar]
  29. Raymond M., Gros P., Whiteway M., Thomas D. Y. Functional complementation of yeast ste6 by a mammalian multidrug resistance mdr gene. Science. 1992 Apr 10;256(5054):232–234. doi: 10.1126/science.1348873. [DOI] [PubMed] [Google Scholar]
  30. Rosenberg M. F., Callaghan R., Ford R. C., Higgins C. F. Structure of the multidrug resistance P-glycoprotein to 2.5 nm resolution determined by electron microscopy and image analysis. J Biol Chem. 1997 Apr 18;272(16):10685–10694. doi: 10.1074/jbc.272.16.10685. [DOI] [PubMed] [Google Scholar]
  31. Sarkadi B., Müller M., Homolya L., Holló Z., Seprödi J., Germann U. A., Gottesman M. M., Price E. M., Boucher R. C. Interaction of bioactive hydrophobic peptides with the human multidrug transporter. FASEB J. 1994 Jul;8(10):766–770. doi: 10.1096/fasebj.8.10.7914178. [DOI] [PubMed] [Google Scholar]
  32. Shapiro A. B., Ling V. ATPase activity of purified and reconstituted P-glycoprotein from Chinese hamster ovary cells. J Biol Chem. 1994 Feb 4;269(5):3745–3754. [PubMed] [Google Scholar]
  33. Shapiro A. B., Ling V. Reconstitution of drug transport by purified P-glycoprotein. J Biol Chem. 1995 Jul 7;270(27):16167–16175. doi: 10.1074/jbc.270.27.16167. [DOI] [PubMed] [Google Scholar]
  34. Sharma R. C., Inoue S., Roitelman J., Schimke R. T., Simoni R. D. Peptide transport by the multidrug resistance pump. J Biol Chem. 1992 Mar 25;267(9):5731–5734. [PubMed] [Google Scholar]
  35. Sharom F. J., DiDiodato G., Yu X., Ashbourne K. J. Interaction of the P-glycoprotein multidrug transporter with peptides and ionophores. J Biol Chem. 1995 Apr 28;270(17):10334–10341. doi: 10.1074/jbc.270.17.10334. [DOI] [PubMed] [Google Scholar]
  36. Sharom F. J. The P-glycoprotein efflux pump: how does it transport drugs? J Membr Biol. 1997 Dec 1;160(3):161–175. doi: 10.1007/s002329900305. [DOI] [PubMed] [Google Scholar]
  37. Sharom F. J., Yu X., Chu J. W., Doige C. A. Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells. Biochem J. 1995 Jun 1;308(Pt 2):381–390. doi: 10.1042/bj3080381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sharom F. J., Yu X., DiDiodato G., Chu J. W. Synthetic hydrophobic peptides are substrates for P-glycoprotein and stimulate drug transport. Biochem J. 1996 Dec 1;320(Pt 2):421–428. doi: 10.1042/bj3200421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sharom F. J., Yu X., Doige C. A. Functional reconstitution of drug transport and ATPase activity in proteoliposomes containing partially purified P-glycoprotein. J Biol Chem. 1993 Nov 15;268(32):24197–24202. [PubMed] [Google Scholar]
  40. Toppmeyer D. L., Slapak C. A., Croop J., Kufe D. W. Role of P-glycoprotein in dolastatin 10 resistance. Biochem Pharmacol. 1994 Aug 3;48(3):609–612. doi: 10.1016/0006-2952(94)90292-5. [DOI] [PubMed] [Google Scholar]
  41. Twentyman P. R. Cyclosporins as drug resistance modifiers. Biochem Pharmacol. 1992 Jan 9;43(1):109–117. doi: 10.1016/0006-2952(92)90668-9. [DOI] [PubMed] [Google Scholar]
  42. Twentyman P. R., Luscombe M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer. 1987 Sep;56(3):279–285. doi: 10.1038/bjc.1987.190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Urbatsch I. L., al-Shawi M. K., Senior A. E. Characterization of the ATPase activity of purified Chinese hamster P-glycoprotein. Biochemistry. 1994 Jun 14;33(23):7069–7076. doi: 10.1021/bi00189a008. [DOI] [PubMed] [Google Scholar]
  44. Zhang L., Sachs C. W., Fine R. L., Casey P. J. Interaction of prenylcysteine methyl esters with the multidrug resistance transporter. J Biol Chem. 1994 Jun 10;269(23):15973–15976. [PubMed] [Google Scholar]

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