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A high-throughput assay for assessing the cell permeability of combinatorial libraries

Nature Biotechnology volume 23, pages 746–751 (2005)Cite this article

Abstract

There is great interest in the identification of synthetic molecules that are capable of manipulating protein-protein interactions in living cells. Peptides, unlike other classes of small molecules, have binding properties appropriate for this application, but most are poorly cell permeable and sensitive to proteases. Therefore, considerable effort has been expended in the development of libraries of oligomeric peptide-like molecules1,2,3,4. However, there are no clear-cut rules to guide the design of libraries rich in cell permeable compounds. Furthermore, currently available empirical methods to assess permeability may not accurately reflect true permeability and/or are capable of only modest throughput5,6,7,8,9,10,11,12. We describe here an assay for assessing the relative cell permeability of synthetic molecules in the context of steroid fusions that is capable of high throughput and can be used in any transfectable cell line.

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Figure 1: Illustration of the design of the cell permeability assay.
Figure 2: Validation of the cell permeability assay.
Figure 3: Determination of the cell permeability of library-derived peptoids using a high-throughput assay.
Figure 4: The dependence of the maximum induction of luciferase on the net charges carried by the OxDex-AEEA-tetrameric peptoid-βAla conjugates.

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Acknowledgements

We thank John Shelton of the Department of Internal Medicine and Kausar Nadim of the Center for Biomedical Inventions in the UT-Southwestern Medical Center for help with the fluorescence imaging. This work was supported by grants from the National Institutes of Health (P01-DK58398) and the Welch Foundation (I-1299).

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Author notes

  1. Peng Yu and Bo Liu: These authors contributed equally to this work.

Authors and Affiliations

  1. Departments of Internal Medicine and Molecular Biology and the Center for Biomedical Inventions, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, 75390-8573, Texas, USA

    Peng Yu, Bo Liu & Thomas Kodadek

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  1. Peng Yu
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Correspondence to Thomas Kodadek.

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Supplementary information

Supplementary Fig. 1

Molecular structures of dexamethasone and variousderivatives used in this study. (PDF 700 kb)

Supplementary Fig. 2

Validation of the high throughput assay. (PDF 372 kb)

Supplementary Fig. 3

Evaluation of the cell permeability of the peptoids byfluorescence imaging. (PDF 608 kb)

Supplementary Table 1

Summary of the cell permeability of tetrameric peptoids with different monomer compositions in the high throughput assay. (PDF 54 kb)

Supplementary Table 2

Summary of the parameters from cell permeability and in vitro competition assay for OxDex-tetrameric peptoids carrying different net charges at neutral pH. (PDF 49 kb)

Supplementary Methods (PDF 116 kb)

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Yu, P., Liu, B. & Kodadek, T. A high-throughput assay for assessing the cell permeability of combinatorial libraries. Nat Biotechnol 23, 746–751 (2005). https://doi.org/10.1038/nbt1099

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