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Published May 18, 2023 | Supplemental Material + Published
Journal Article Open

Stereochemical engineering yields a multifunctional peptide macrocycle inhibitor of Akt2 by fine-tuning macrocycle-cell membrane interactions


Macrocycle peptides are promising constructs for imaging and inhibiting extracellular, and cell membrane proteins, but their use for targeting intracellular proteins is typically limited by poor cell penetration. We report the development of a cell-penetrant high-affinity peptide ligand targeted to the phosphorylated Ser474 epitope of the (active) Akt2 kinase. This peptide can function as an allosteric inhibitor, an immunoprecipitation reagent, and a live cell immunohistochemical staining reagent. Two cell penetrant stereoisomers were prepared and shown to exhibit similar target binding affinities and hydrophobic character but 2-3-fold different rates of cell penetration. Experimental and computational studies resolved that the ligands' difference in cell penetration could be assigned to their differential interactions with cholesterol in the membrane. These results expand the tool kit for designing new chiral-based cell-penetrant ligands.

Additional Information

© The Author(s) 2023. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. The various PCCs reported here were developed under funding from the National Cancer Institute through grant #1U54 CA199090-01 (JRH PI) and the WA State Andy Hill CARE Foundation. WAG and AM thank NIH (NIH R35HL150807) for support. Contributions. A.N. and S.D. designed the study under the guidance of J.R.H. Experiments were performed by A.N., S.D., M.B.Y, B.A.-V., and data were analyzed by A.N. and S.D. Molecular dynamics were performed by A.M., under the guidance of W.A.G. Molecular dynamics conformational analysis was performed by A.N., and S.-K.K. Flow cytometry data were analyzed by A.N and Y.S. The manuscript was written by A.N., A.M., J.R.H., and W.A.G. All authors have approved the final version. Data availability. All data generated or analyzed during this study are included in this published article (and its supplementary information files). Competing interests. J.R.H. is a consultant to Regeneron, which is a company seeking to commercialize PCC technology. All other authors declare no competing interests.

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Supplemental Material - 42004_2023_890_MOESM1_ESM.pdf


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August 22, 2023
October 20, 2023