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Published December 10, 2021 | Submitted + Supplemental Material
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Stereochemical engineering of a peptide macrocycle allosteric inhibitor of phospho-Akt2 controls cell penetration by fine-tuning macrocycle-cell membrane interactions


We report the development of a cell-penetrant cyclic loop biligand that selectively binds, in vitro, to the phosphorylated Ser474 site of Protein Kinase B (p-Akt2) with high affinity (K_D = 10 nM). The cyclic loop biligand consists of a linear peptide joined to a macrocycle peptide through triazole linkage, and it was isolated through two iterative in situ screens. This biligand allosterically inhibited kinase activity of Akt2 but it was cell-impermeable, as isolated from the screening process. Since Akt2 is an oncoprotein hyperactivated via phosphorylation at Ser474 in cancers, we sought to visualize p-Akt2 in live cancer cells using the developed biligand. To this end, we matured this biligand into a cell-penetrant reagent through systematic iterations of its chemical structure to promote cell-penetrating properties, while retaining its binding and inhibition for p-Akt2. Two retro-inverso, N-methylated versions of the macrocyclic ligand were developed which were uptaken by live cancer cells, while retaining their high affinities for pAkt2. Interestingly, the stereochemistry of two amino acid residues in the cell-penetrant ligands exhibited strong influence on their extent of cell penetration. This phenomenon of difference in cell penetration was explored through metadynamics simulations of each ligand in the cell membrane. It was found that the ligand uptaken to a greater extent by cells had more intramolecular interactions with itself and had fewer cholesterol molecules associated with it, which aided in its cell-penetration.

Additional Information

The content is available under CC BY NC ND 4.0 License. The various PCCs reported here were developed under funding from the National Cancer Institute through grant #1U54 CA199090-01 (JRH PI). WAG and AM thank NIH (NIH R35HL150807) for support. Author's competing interest statement. James R Heath is a founder and board member of Indi Molecular, which is a company seeking to commercialize PCC technology. The author(s) declare that they have sought and gained approval from the relevant ethics committee/IRB for this research and its publication.

Attached Files

Submitted - 10.26434_chemrxiv-2021-kldh7.pdf

Supplemental Material - supplementary-information.pdf


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August 20, 2023
October 23, 2023