α/β-Peptide Foldamers Targeting Intracellular Protein-Protein Interactions with Activity in Living Cells

Abstract

Peptides can be developed as effective antagonists of protein-protein interactions, but conventional peptides (i.e., oligomers of l-α-amino acids) suffer from significant limitations in vivo. Short half-lives due to rapid proteolytic degradation and an inability to cross cell membranes often preclude biological applications of peptides. Oligomers that contain both α- and β-amino acid residues ("α/β-peptides") manifest decreased susceptibility to proteolytic degradation, and when properly designed these unnatural oligomers can mimic the protein-recognition properties of analogous "α-peptides". This report documents an extension of the α/β-peptide approach to target intracellular protein-protein interactions. Specifically, we have generated α/β-peptides based on a "stapled" Bim BH3 α-peptide, which contains a hydrocarbon cross-link to enhance α-helix stability. We show that a stapled α/β-peptide can structurally and functionally mimic the parent stapled α-peptide in its ability to enter certain types of cells and block protein-protein interactions associated with apoptotic signaling. However, the α/β-peptide is nearly 100-fold more resistant to proteolysis than is the parent stapled α-peptide. These results show that backbone modification, a strategy that has received relatively little attention in terms of peptide engineering for biomedical applications, can be combined with more commonly deployed peripheral modifications such as side chain cross-linking to produce synergistic benefits.

Figure 1

(A) Primary sequences of α- and α/β-peptides used in this study. The crosslinked α-peptide α-1 has been referred to as BimSAHB in prior reports.15,16 Non-natural amino acid residues are indicated by colored circles: green for S5 residue used for crosslinking, and orange for cyclic β-residues. A horizontal line connecting two S5 residues indicates that these residues have been crosslinked using olefin metathesis. (B) Structures of a generic α-residue, the S5 residue (*), two crosslinked S5 residues, and the cyclic β-residues ACPC (X) and sAPC (U).

Figure 2

Far-UV circular dichroism spectra of α- and α/β-peptides at 50 μM in water at 20 °C.

Figure 3

Crystal structures of α/β-peptides α/β-1 and α/β-1-LIN bound to Bcl-2. (A) Structure of α/β-1 bound to Bcl-2 (PDB: 5AGW). (B) Structure of α/β-1-LIN bound to Bcl-2 (PDB: 5AGX). In A and B, the α/β-peptide is colored by residue type (yellow for natural α-residues, green for S5 residues, and orange for cyclic β-residues) and the Bcl-2 surface is shown in gray. (C) Overlay of α/β-1 and α/β-1-LIN with previously reported structures of an analogue of α-Bim (bound to Bcl-x_L, PDB: 3FDL) and an analogue of α-1 (bound to Bcl-x_L, PDB: 2YQ6). Positions of key protein-contacting hydrophobic residues h1-h4 and the position of the crosslink (for α-1 and α/β-1) are labeled.

Figure 4

Cytochrome c release assay on permeabilized wild-type (wt) or bax^−/−/bak^−/− mouse embryonic fibroblasts (MEFs) treated with 10 μM of the indicated α- or α/β-peptide for 1 hour. α/β-1, α/β-2, and α-Bim were able to induce cytochrome c release from the mitochondria-containing pellet fraction (P) to the soluble cytosolic fraction (S) in wt MEFs, but not in bax^−/−/bak^−/− MEFs, as indicated by western blot for cytochrome c. No cytochrome c release was seen for negative control Bim4E (DMRPEIWEAQEERREGDEENAYYARR-OH). The lower panel shows western blot for Bak as a control for membrane permeabilization. Bak is a mitochondrial protein and therefore should not appear in the cytosolic fraction. bax-^−/−/bak^−/− MEFs do not have Bak, and therefore no band is apparent. Irrelevant intervening lanes between α/β-1 and α/β-2 have been cropped from the image.

Figure 5

Viability of various cell lines (relative to DMSO control) when exposed to 10 μM α-1 (black bars) or α/β-1 (white bars) for 24 hours, as measured using CellTiter-Glo assay. Each bar represents the mean ± SD for two independent experiments. [Although the data seem clear, I wonder if we should give some indication of statistical significance. Specifically, can we state or indicate with symbols that for all cells OTHER THAN U937, growth in the presence of peptides is statistically indistinguishable from a "no treatment" control? In terms of layout, can we should move U937 next to wtMEF, or somewhere in the middle, to make it clearer that there is one cell line that is susceptible? It is easy to miss this information with U937 at the end, since there are no “bars”.]

Figure 6

Viability of U937 cells (relative to DMSO control) when treated with various concentrations of α- or α/β-peptides in the absence (red) or presence (blue) of 50 μM caspase inhibitor Q-VD-OPh (QVD), as determined by CellTiter-Glo assay. Cells were treated with the indicated α- or α/β-peptide for 2 hours under serum-free conditions, followed by the addition of serum-containing media and additional incubation until the indicated time point. Each data point represents the mean ± SEM for 3-5 replicate measurements.

Figure 7

(A) Analysis of U937 cell killing over time induced by 5 μM α/β-1 or α-1 in the absence or presence of 50 μM QVD-OPh (QVD). Green cells indicate those stained for CellTracker Green dye (living cells). Red cells indicate cells stained by propidium iodide (dead cells). (B) Close up of the indicated conditions from panel A, highlighting morphological changes resulting from treatment with α/β-1 relative to DMSO control.

Figure 8

Cytochrome c release assay on unpermeabilized U937 cells treated with 10 μM of the indicated α- or α/β-peptide for 2 hours. α/β-1, but not α/β-2 or α-Bim, was able to induce cytochrome c release from the mitochondria-containing pellet fraction (P) to the soluble cytosolic fraction (S). The lower panel indicates western blot for Bak as a control for membrane permeabilization.

Figure 9

Analysis of U937 cellular uptake of fluorescein (Flu)-labeled analogues of α- and α/β-peptides by live-cell confocal microscopy. Cells were treated with 2 μM of the indicated α- or α/β-peptide in serum-free media for 3 hours prior to imaging. Images are obtained under 90× magnification, showing the channels for green (fluorescein, peptide), red (WGA488, outer membrane stain), and blue (Hoescht 33342, nuclear stain). Scale bars represent 25 μm.

Figure 10

Analysis of MEF cellular uptake of fluorescein (Flu)-labeled analogues of α- and α/β-peptides by live-cell confocal microscopy. Cells were treated with 2 μM of the indicated α- or α/β-peptide in serum-free media for 3 hours prior to imaging. Images are obtained under 60× magnification, showing the channels for green (fluorescein, peptide), red (WGA488, outer membrane stain), and blue (Hoescht 33342, nuclear stain). Scale bars represent 25 μm.