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Small-molecule mimics of an α-helix for efficient transport of proteins into cells

Nature Methods volume 4, pages 153–159 (2007)Cite this article

Abstract

We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an example of a protein cargo, we applied this new technology to the internalization of the DNA replication licensing repressor geminin, in vitro, providing evidence that extracellularly delivered SMoC-geminin can have an antiproliferative effect on human cancer cells. Uptake of SMoC-geminin was inhibited at 4 °C and by chlorpromazine, a compound that induces misassembly of clathrin-coated pits at the cell surface. Thus the mechanism of uptake is likely to be clathrin-mediated endocytosis.

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Figure 1: Molecular modeling and chemistry of SMoCs.
Figure 2: SMoCs are rapid and efficient transporters of FITC in primary and cultured cells.
Figure 3: Coupling of geminin to SMoC allows rapid transportation of protein into cells.
Figure 4: Influence of chemical inhibitors and temperature on uptake of SMoCs with different cargo.
Figure 5: The biological activity of protein cargo is not affected by SMoC-mediated delivery.

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References

  1. Bonetta, L. Getting proteins protein into cells. Scientist 16, 38–40 (2002).

    Google Scholar 

  2. Roberts, J.P. Buyer's guide to protein transduction reagents - A range of chemicals and peptides are available to help get proteins into cells. Scientist 18, 42–43 (2004).

    Google Scholar 

  3. Wadia, J.S. & Dowdy, S.F. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv. Drug Deliv. Rev. 57, 579–596 (2005).

    Article  CAS  Google Scholar 

  4. Joliot, A. & Prochiantz, A. Transduction peptides: from technology to physiology. Nat. Cell Biol. 6, 189–196 (2004).

    Article  CAS  Google Scholar 

  5. Morris, M.C., Depollier, J., Mery, J., Heitz, F. & Divita, G. A peptide carrier for the delivery of biologically active proteins into mammalian cells. Nat. Biotechnol. 19, 1173–1176 (2001).

    Article  CAS  Google Scholar 

  6. Schwarze, S.R., Ho, A., Vocero-Akbani, A. & Dowdy, S.F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science 285, 1569–1572 (1999).

    Article  CAS  Google Scholar 

  7. Li, T., Fan, Y., Luo, Y., Xiao, B. & Lu, C. In vivo delivery of a XIAP (BIR3-RING) fusion protein containing the protein transduction domain protects against neuronal death induced by seizures. Exp. Neurol. 197, 301–308 (2006).

    Article  CAS  Google Scholar 

  8. Choi, H.S. et al. Transduced Tat-α-synuclein protects against oxidative stress in vitro and in vivo. J. Biochem. Mol. Biol. 39, 253–262 (2006).

    CAS  PubMed  Google Scholar 

  9. Cashman, S.M., Morris, D.J. & Kumar-Singh, R. Evidence of protein transduction but not intercellular transport by proteins fused to HIV tat in retinal cell culture and in vivo. Mol. Ther. 8, 130–142 (2003).

    Article  CAS  Google Scholar 

  10. McGarry, T.J. & Kirschner, M.W. Geminin, an inhibitor of DNA replication, is degraded during mitosis. Cell 93, 1043–1053 (1998).

    Article  CAS  Google Scholar 

  11. Magzoub, M., Eriksson, L.E. & Graslund, A. Conformational states of the cell-penetrating peptide penetratin when interacting with phospholipid vesicles: effects of surface charge and peptide concentration. Biochim. Biophys. Acta 1563, 53–63 (2002).

    Article  CAS  Google Scholar 

  12. Futaki, S. et al. Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery. J. Biol. Chem. 276, 5836–5840 (2001).

    Article  CAS  Google Scholar 

  13. Shibata, A. et al. Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes. Biochim. Biophys. Acta 1616, 147–155 (2003).

    Article  CAS  Google Scholar 

  14. Ho, A., Schwarze, S.R., Mermelstein, S.J., Waksman, G. & Dowdy, S.F. Synthetic protein transduction domains: enhanced transduction potential in vitro and in vivo. Cancer Res. 61, 474–477 (2001).

    CAS  PubMed  Google Scholar 

  15. Orner, B.P., Ernst, J.T. & Hamilton, A.D. Toward proteomimetics: terphenyl derivatives as structural and functional mimics of extended regions of an α-helix. J. Am. Chem. Soc. 123, 5382–5383 (2001).

    Article  CAS  Google Scholar 

  16. Kutzki, O. et al. Development of a potent Bcl-x(L) antagonist based on α-helix mimicry. J. Am. Chem. Soc. 124, 11838–11839 (2002).

    Article  CAS  Google Scholar 

  17. Jacoby, E. Biphenyls as potential mimetics of protein α-helix. Bioorg. Med. Chem. Lett. 12, 891–893 (2002).

    Article  CAS  Google Scholar 

  18. Rothbard, J.B., Jessop, T.C. & Wender, P.A. Adaptive translocation: the role of hydrogen bonding and membrane potential in the uptake of guanidinium-rich transporters into cells. Adv. Drug Deliv. Rev. 57, 495–504 (2005).

    Article  CAS  Google Scholar 

  19. Wohlschlegel, J.A. et al. Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290, 2309–2312 (2000).

    Article  CAS  Google Scholar 

  20. Okorokov, A.L. et al. Molecular structure of human geminin. Nat. Struct. Mol. Biol. 11, 1021–1022 (2004).

    Article  CAS  Google Scholar 

  21. Nunn, C.M. et al. Crystal structure of tobacco etch virus protease shows the protein C terminus bound within the active site. J. Mol. Biol. 350, 145–155 (2005).

    Article  CAS  Google Scholar 

  22. Kulartz, M. & Knippers, R. The replicative regulator protein geminin on chromatin in the HeLa cell cycle. J. Biol. Chem. 279, 41686–41694 (2004).

    Article  CAS  Google Scholar 

  23. Wang, L.H., Rothberg, K.G. & Anderson, R.G. Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. J. Cell Biol. 123, 1107–1117 (1993).

    Article  CAS  Google Scholar 

  24. Rothberg, K.G. et al. Caveolin, a protein component of caveolae membrane coats. Cell 68, 673–682 (1992).

    Article  CAS  Google Scholar 

  25. West, M.A., Bretscher, M.S. & Watts, C. Distinct endocytotic pathways in epidermal growth factor-stimulated human carcinoma A431 cells. J. Cell Biol. 109, 2731–2739 (1989).

    Article  CAS  Google Scholar 

  26. Sampath, P. & Pollard, T.D. Effects of cytochalasin, phalloidin, and pH on the elongation of actin filaments. Biochemistry 30, 1973–1980 (1991).

    Article  CAS  Google Scholar 

  27. Murray, J.W. & Wolkoff, A.W. Roles of the cytoskeleton and motor proteins in endocytic sorting. Adv. Drug Deliv. Rev. 55, 1385–1403 (2003).

    Article  CAS  Google Scholar 

  28. Stoeber, K. et al. Cdc6 protein causes premature entry into S phase in a mammalian cell-free system. EMBO J. 17, 7219–7229 (1998).

    Article  CAS  Google Scholar 

  29. Kingsbury, S.R. et al. Repression of DNA replication licensing in quiescence is independent of geminin and may define the cell cycle state of progenitor cells. Exp. Cell Res. 309, 56–67 (2005).

    Article  CAS  Google Scholar 

  30. Richard, J.P. et al. Cellular uptake of unconjugated TAT peptide involves clathrin-dependent endocytosis and heparan sulfate receptors. J. Biol. Chem. 280, 15300–15306 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Mitsubishi Pharma Corporation for a visiting fellowship for M.O. and funding for C.V.; HL was funded by the Association for International Cancer Research. We are grateful to A. Okorokov for advice on the generation of geminin loss-of-function mutants and the ΔNt-geminin construct, and to S. Djordjevic for the gift of pRET 3a-TEV plasmid. We thank A. Stuart for advice on inhibitors of pathways of endocytosis and H. Engel for technical assistance. We thank A. Farshchi and S. Paige for technical assistance with site-directed mutagenesis and protein expression. We are grateful to L. Rakes and L. Suarez-Delgado for assistance with cell-proliferation assays. The geminin studies were funded by Cancer Research UK Programme Grant C428/A6263. S.R.K. is supported by an MRC Research Studentship.

Author information

Author notes

  1. Heike Laman

    Present address: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QP, UK

  2. Kathryn Leigh Eward

    Present address: Department of Life Sciences, Faculty of Science and Technology, Anglia Ruskin University, East Road, Cambridge, CB1 1PT, UK

  3. Masahiro Okuyama, Heike Laman and Sarah R Kingsbury: These authors contributed equally to this work.

Authors and Affiliations

  1. Biological and Medicinal Chemistry Group, Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, UK

    Masahiro Okuyama, Cristina Visintin & David L Selwood

  2. Cancer Research UK Viral Oncology Group, Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, UK

    Heike Laman & Chris Boshoff

  3. Cancer Research UK Chromosomal Replication Group, Wolfson Institute for Biomedical Research, University College London, Gower Street, London, WC1E 6BT, UK

    Sarah R Kingsbury, Elisabetta Leo, Kathryn Leigh Eward, Kai Stoeber & Gareth H Williams

  4. Department of Pathology, Royal Free and University College Medical School, University College London, London, WC1E 6JJ, UK

    Kai Stoeber & Gareth H Williams

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  1. Masahiro Okuyama
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Contributions

M.O. and D.L.S., chemical design and molecular modeling; M.O. and C.V. chemical synthesis; H.L., C.B. and E.L., studies on transport of dyes and SMoCs into cells; H.L. and E.L., studies on mechanism of uptake; S.R.K., K.L.E., K.S. and G.H.W., coupling of geminin to SMoCs and studies on antiproliferative action of SMoC geminin.

Corresponding authors

Correspondence to Gareth H Williams or David L Selwood.

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Competing interests

M.O. was funded as a visiting researcher by Mitsubishi Pharma, and C.V. was funded by Mitsubishi Pharma for one year. The university has filed a patent, WO2005123676(A1), on this work. The authors may eventually received financial reward through a university revenue sharing scheme.

Supplementary information

Supplementary Fig. 1

Influence of chemical inhibitors on uptake of SMoC.

Supplementary Fig. 2

Inhibition of G0-S progression with SMoC-geminins.

Supplementary Methods

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Okuyama, M., Laman, H., Kingsbury, S. et al. Small-molecule mimics of an α-helix for efficient transport of proteins into cells. Nat Methods 4, 153–159 (2007). https://doi.org/10.1038/nmeth997

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