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Biophysical determinants for cellular uptake of hydrocarbon-stapled peptide helices

Nature Chemical Biology volume 12, pages 845–852 (2016)Cite this article

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Abstract

Hydrocarbon-stapled peptides are a class of bioactive alpha-helical ligands developed to dissect and target protein interactions. While there is consensus that stapled peptides can be effective chemical tools for investigating protein regulation, their broader utility for therapeutic modulation of intracellular interactions remains an active area of study. In particular, the design principles for generating cell-permeable stapled peptides are empiric, yet consistent intracellular access is essential to in vivo application. Here, we used an unbiased statistical approach to determine which biophysical parameters dictate the uptake of stapled-peptide libraries. We found that staple placement at the amphipathic boundary combined with optimal hydrophobic and helical content are the key drivers of cellular uptake, whereas excess hydrophobicity and positive charge at isolated amino acid positions can trigger membrane lysis at elevated peptide dosing. Our results provide a design roadmap for maximizing the potential to generate cell-permeable stapled peptides with on-mechanism cellular activity.

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Figure 1: Microscopy-based quantitation of stapled peptide uptake.
Figure 2: Determinants of cellular uptake for differentially-stapled BIM BH3 peptides.
Figure 3: Impact of point mutagenesis on the cellular uptake of BIM SAHBA1.
Figure 4: Target binding affinity and cytotoxicity of stapled BIM BH3 libraries.
Figure 5: Determinants of cellular lysis for stapled BIM BH3 peptides.
Figure 6: Identification of lead stapled peptide constructs for cellular and in vivo application.

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Acknowledgements

We thank E. Smith for graphics support, S. Rudnicki of the Institute of Chemistry and Cell Biology–Longwood for assistance with IXM screening and analysis, and J. Opferman of St. Jude Children's Research Hospital for the BCL-XL-reconstituted p185+Arf−/−Mcl-1-deleted B-ALL cells. This research was supported by NIH grants 1R35CA197583 and 1R21CA209358, a Leukemia and Lymphoma Society (LLS) Marshall A. Lichtman Specialized Center of Research project grant, the William Lawrence and Blanche Hughes Foundation, the Todd J. Schwartz Memorial Fund, the Wolpoff Family Foundation, and an LLS Scholar Award to L.D.W. E.M. and D.S.N. are supported in part by the Dana–Farber/Harvard Cancer Center Support grant 5P30CA006516.

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Authors and Affiliations

  1. Department of Pediatric Oncology, Linde Program in Cancer Chemical Biology, Dana–Farber Cancer Institute, Boston, Massachusetts, USA

    Gregory H Bird, Kwadwo Opoku-Nsiah, Margaret A Lammert, Marina Godes & Loren D Walensky

  2. Department of Biostatistics and Computational Biology, Dana–Farber Cancer Institute, Boston, Massachusetts, USA

    Emanuele Mazzola & Donna S Neuberg

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  1. Gregory H Bird
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Contributions

G.H.B., E.M., D.S.N., and L.D.W. designed the study; G.H.B., K.O.-N., M.G., M.A.L. and L.D.W. generated stapled peptides, performed the cellular-uptake experiments, and conducted binding and cell-viability analyses; E.M. and D.S.N. performed the statistical analyses; G.H.B., E.M., D.S.N., and L.D.W. analyzed the data and wrote the manuscript, which was reviewed by all co-authors.

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Correspondence to Loren D Walensky.

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L.D.W. is a scientific advisory board member and consultant for Aileron Therapeutics.

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Bird, G., Mazzola, E., Opoku-Nsiah, K. et al. Biophysical determinants for cellular uptake of hydrocarbon-stapled peptide helices. Nat Chem Biol 12, 845–852 (2016). https://doi.org/10.1038/nchembio.2153

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