Bridged beta(3)-peptide inhibitors of p53-hDM2 complexation: correlation between affinity and cell permeability

Abstract

Beta-peptides possess several features that are desirable in peptidomimetics; they are easily synthesized, fold into stable secondary structures in physiologic buffers, and resist proteolysis. They can also bind to a diverse array of proteins to inhibit their interactions with alpha-helical ligands. beta-peptides are usually not cell-permeable, however, and this feature limits their utility as research tools and potential therapeutics. Appending an Arg(8) sequence to a beta-peptide improves uptake but adds considerable mass. We previously reported that embedding a small cationic patch within a PPII, alpha-, or beta-peptide helix improves uptake without the addition of significant mass. In another mass-neutral strategy, Verdine, Walensky, and others have reported that insertion of a hydrocarbon bridge between the i and i + 4 positions of an alpha-helix also increases cell uptake. Here we describe a series of beta-peptides containing diether and hydrocarbon bridges and compare them on the basis of cell uptake and localization, affinities for hDM2, and 14-helix structure. Our results highlight the relative merits of the cationic-patch and hydrophobic-bridge strategies for improving beta-peptide uptake and identify a surprising correlation between uptake efficiency and hDM2 affinity.

Figure 1

Helical net representation of β-peptides studied herein. β³-homoamino acids are identified by the single-letter code used for the corresponding α–amino acid. Orn represents ornithine. Z represents 3-(S)-3-amino-4-(2-trifluoromethylphenyl)-butyric acid.

Figure 2

CD analysis of β-peptides containing hydrocarbon or diether bridges between residues (A) 2 and 5 or (B) 4 and 7. Fluorescence polarization (FP) analysis of hDM2 binding by β-peptides containing (C) hydrocarbon or (D) diether bridges.

Figure 3

Computational model of hDM2 (grey) in complex with (A) 25.C-s or (B) 47.C-s.

Figure 4

HeLa cell uptake and localization of Flu-labeled β-peptides. (A,B) HeLa cells were incubated with 2 μM β–peptide for 4 h, treated with 0.25% trypsin for 10 min, washed with cold DMEM and PBS, and analyzed using flow cytometry. (C) Confocal microscopy of HeLa cells treated with 20 μM of the indicated β-peptide (green), 5 mg•mL^-1 Alexa Fluor 647-transferrin (red) and 150 nM Hoescht 33342 (blue).