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 crosslink 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 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 crosslinking to produce synergistic benefits.

An α‐Helix‐Mimicking 12,13‐Helix: Designed α/β/γ‐Foldamers as Selective Inhibitors of Protein–Protein Interactions

“…Amongst a multitude of foldamer classes where structural/ conformational determinants have been mapped,1, 2, 3, 4 β‐peptides and hybrid α/β‐peptides, in which β‐amino acids are dispersed along an α‐peptide backbone, can inhibit α‐helix‐mediated protein–protein interactions22, 23, 24, 25, 26, 27, 28, 29 and mimic the structure and the function of protein surfaces 30, 31. Nonetheless foldamers that more accurately mimic the topology and topography of the α‐helix might prove advantageous in comparison to β‐ and α/β‐peptides, which may not fully mimic the spatial presentation of α‐helix side chains.…”

An α‐Helix‐Mimicking 12,13‐Helix: Designed α/β/γ‐Foldamers as Selective Inhibitors of Protein–Protein Interactions

“…Peptides 1-8 were subjected to ab initio geometry optimization by DFT using Gaussian09 at the B3LYP/6-311G(d,p) level of theory. Thel owest-energy structures of 1-4 and 5-8 were superimposed (Figure 3a,b) and the backbone of peptide 2 (as ar epresentative example) was overlaid with the crystal structure of p53 [16][17][18][19][20][21][22][23][24][25][26][27][28][29] (Figure 3c)u sing g 4 -amino acid C(a) atoms as the basis of the alignment. Thesuperimposition gave an excellent RMSD value of around 0.9 (Figure 3d,e), thus strongly suggesting that the a/b/g-peptides 1-8 effectively mimic an a-helix.…”

“…Thep roteolytic stability of a/b/g-peptides 1-8 was investigated using a-chymotrypsin (a-CT) as ar epresentative protease,s ince this enzyme selectively hydrolyses the amide bond on the C-terminal side of hydrophobic residues such as Leu, Tr p, and Phe,a ll of which are present in the a/b/gpeptide sequences.T he p53 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] peptide was also studied for comparison (see the Supporting Information). Using HPLC to assess the progress of peptide digestion, it transpired that all eight a/b/g-peptides displayed considerably greater resistance to a-CT than did the native p53 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] (!…”

“…Using HPLC to assess the progress of peptide digestion, it transpired that all eight a/b/g-peptides displayed considerably greater resistance to a-CT than did the native p53 [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] (! 32-fold for 2, 4, 6, and 8 and !…”

“…[17][18][19][20][21] However,t here is still aneed to develop ligands that more effectively mimic the conformation and molecular recognition capabilities of the ahelix. Herein, we present the bottom-up design of hybrid a/b/ g-peptides that assume an a-helix-mimicking 12,13-helical conformation and function as effective inhibitors of the p53/ hDM2 interaction.Amongst am ultitude of foldamer classes where structural/ conformational determinants have been mapped, [1][2][3][4] bpeptides and hybrid a/b-peptides,inwhich b-amino acids are dispersed along an a-peptide backbone,c an inhibit a-helixmediated protein-protein interactions [22][23][24][25][26][27][28][29] and mimic the structure and the function of protein surfaces. [30,31] Nonetheless foldamers that more accurately mimic the topology and topography of the a-helix might prove advantageous in comparison to b-and a/b-peptides,which may not fully mimic the spatial presentation of a-helix side chains.S everal foldamer scaffolds have been hypothesized to have potential for the inhibition of a-helix-mediated PPIs, [32][33][34][35] but they have not yet been shown to do so experimentally.…”