Cell-permeable beta-peptide inhibitors of p53/hDM2 complexation

Abstract

Look at what the cat(ionic motif) dragged in! We report a general strategy to increase the cell permeability of beta(3)-peptides. Introduction of a minimal cationic motif within the folded structure of a high-affinity beta(3)-peptide ligand for hDM2 led to molecules with high 3(14)-helical structure, high hDM2 affinity and sufficient cell permeability to upregulate p53-dependent genes in live mammalian cells. Minimally cationic beta(3)-peptides represent the critical first step towards a class of protease-resistant peptidomimetics that might modulate intracellular biological pathways.

Figure 1

(a) Two strategies to increase the cell permeability of β³-peptides that bind hDM2. In Strategy 1, β³-homoarginine residues are embedded into the β³-peptide structural face, while in Strategy 2 they are embedded into the salt bridge face. (b) Helical net representation of β³-peptides studied as controls. β³-homoamino acids are identified by the one-letter-code corresponding to the analogous β³-amino acid where CF₃-F denotes 3-trifluoromethylphenylalanine.

Figure 2

Circular dichroism spectra of β³-peptides and controls (50 μM) in Tris buffer (10 mM Tris, 100 mM NaCl, 0.01% Tween, pH 7.4). β53-12 (a) β53-12R₂ , β53-12R₃ , β53-8 ,β53-8R₃ ; (b) β53-12SB₂ , β53-12SB₃ , β53-3SB₃ ; (c) β53-12R₆-1 , β53-12R₆-2 , βNEGR₆ ; (d) β53-12R₈ , β53-3R₈ . [θ]_MRW = mean residue molar ellipticity.

Figure 3

Plots illustrating direct (a-c) and competition (d) fluorescence polarization (FP) analysis of hDM2 binding by β³-peptides studied herein. Equilibrium reactions were performed in Tris buffer (see legend to Figure 2). (a-c): Plots illustrating the observed polarization of the indicated fluorescently labeled β³-peptide as a function of [hDM2]_1-188. β53-12 (a) β53-12R₂ , β53-12R₃ , β53-8 ,β53-8R₃ ; (b) β53-12SB₂ , β53-12SB₃ , β53-3SB₃ ; (c) β53-12R₆-1 , β53-12R₆-2 , βNEGR₆ , β53-12R₈ , β53-3R₈ ; (d): Plot illustrating the observed polarization of the p53AD_15-31^flu complex with hDM2_1-188 as a function of the concentration of unlabeled peptide shown: β53-12, β53-12SB₂, β53-12SB₃, β53-12R₈, and β53-3R₈ (colors as above).

Figure 4

Uptake and viability of β³-peptides studied herein. (A) Flow cytometry analysis of β³-peptide uptake by HCT116 cells after 1 h incubation. Mean cellular fluorescence (MCF) was calculated from the histogram of fluorescence intensity and was corrected for background cellular fluorescence by subtracting the geometric mean of cells treated with only PBS. Each value represents the average of three independent trials. Error bars represent the standard error. (B) Viability of HCT116 cells upon incubation with the indicated β³-peptide (10 μM) for 8 h. Cell viability (CV) was measured using CellTiter-Blue^™ as described in Experimental Methods. Each value represents the average of three independent trials. Error bars represent the standard error.

Figure 5

Confocal microscopy analysis of HCT116 cells treated wtih β³-peptides (a) β53-12^flu, (b) β53-12SB₂^flu, (c) β53-12SB₃^flu. HCT116 cells were incubated with 20 μM fluorescein-labeled β³-peptide (green) for 2 h. Endosomes were visualized using 10 μM 10 kDa dextran labeled with AlexaFluor^™ 647 (red). (a) signal from β³β-peptide only, (b) signal from dextran only, (c) bright-field only, (d) two-color fluorescence with bright-field superposition.

Figure 6

Quantification from Western blot analysis of the effects of β53-12 (dark gray) and β53-12SB₂ (light gray) on p53, hDM2, and p21 levels in HCT116 cells. Original blots in Supporting Information. Chemiluminescent signal was quantified based on pixel intensity using ImgaeQuaNT^™ software. Bar graphs show the percent increase in pixel intensity over control cells without any β³-peptide added (all normalized to GAPDH loading control). Each value represents the average of three independent trials. Error bars represent the standard error.