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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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.
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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.
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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.
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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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DOI: https://doi.org/10.1038/nmeth997
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