Helical β-Peptide Inhibitors of the p53-hDM2 Interaction

Abstract

-Peptides differ from R-peptides by one additional backbone carbon atom and by resistance to metabolism and proteolysis. 1a-c -Peptides fold into helices, sheets, and turns and have wellrecognized potential as peptidomimetics. 2 Two -peptides that interact with discrete macromolecular targets are known: a -tetrapeptide hairpin that binds the somatostatin receptor with nanomolar affinity, 1d,e and a highly cationic 3 -decapeptide that binds TAR RNA. 1f Here, we report a set of 3 -peptides that possess significant 14-helix structure in water; one recognizes a cleft on the surface of the human oncogene product double minute 2 (hDM2) with nanomolar affinity.hDM2 is recognized in vivo by a short R-helix within the activation domain of p53 (p53AD, Figure 1A), the transcription factor that controls cell fate in response to stress. hDM2 negatively regulates p53 function, and disruption of the p53‚hDM2 interaction 3a is an important cancer therapy goal. 3b Three residues on one face of p53AD (F19, W23, and L26) comprise the functional epitope that contributes heavily to the binding energy. 3c-e Modification of a p53AD-based R-peptide with nonnatural R-amino acids that improve helix stability and surface complementarity results in a potent inhibitor that activates apoptosis in vivo. 3f,g The first small molecule inhibitors with submicromolar potency were reported in 2004. 3h Our design began with a 3 -decapeptide with significant 14-helix stability in aqueous solution due to electrostatic macrodipole stabilization and side chain-side chain salt bridges on one helical face. 4 Although the dimensions of a 14-helix differ from those of an R-helix, 2 we hypothesized that the p53AD functional epitope would be recapitulated if the side chains of F19, W23, and L26 were presented at successive positions three residues apart on a stabilized 14-helix ( Figure 1B). Because the 14-helix has almost exactly three residues per turn, these side chains should align upon folding. Four 3 -peptides were designed in which these side chains are appended in both possible orientations on each of the two available 14-helix faces ( 53-1-4, Figure 2).We compared the circular dichroism (CD) spectra of 53-1-4 in aqueous buffer to estimate their 14-helix content ( Figure 3A). 1g-j,2 The 14-helix signature is clearly evident, and the relative minima at 214 nm suggest helical contents between 30% and 50% for 53-1, 3, and 4. 5 Two-dimensional NMR spectroscopy 6 in CD 3 OH confirmed the presence of 14-helix structure in 53-1: ROESY spectra showed four of seven possible C R H(i)fC H(i+3) ROEs and two of six possible C N (i)fC (i+3) ROEs. Additional ROEs may be present but were obscured by resonance overlap; no ROEs inconsistent with 14-helical structure were observed. Analytical ultracentrifugation 5,6 revealed that 53-1, 3, and 4 were monomeric at concentrations between 80 and 400 µM, confirming that these 14-helices are stabilized by intramolecular interactions. We designed a competition fluorescence polarization (FP) assay 7 using hDM2 1-188 (hDM2...

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.…”

Foldameric β‐H18/20P Mixed Helix Stabilized by Head‐to‐Tail Contacts: A Way to Higher‐Order Structures

“…Moreover, the NMR findings support the view that the formation of the large-diameter H18/ 20 P helix is coupled to the head-to-tail association in CD 3 OH. The high-resolution model in Figure 3 reveals that the helix-helix interactions are stabilized by four interchain hydrogen bonds, NH 8 The helical self-association of 4 in CD 3 OH was further investigated by concentration-dependent DOSY NMR spectroscopy. The apparent aggregation number was 3 at a concentration of 100 μm, which rose to 8 at 1 mm (see Figure S6 in the Supporting Information).…”

“…[2,[5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] Although the formation of secondary structures of peptidic foldamers has been thoroughly studied, their higher-order self-organization (tertiary and quaternary structures) and its effects on the folding propensities are less well understood. It has been shown that peptidic foldamers are able to fold cooperatively into helix bundles; [20] β-and α,β-peptide sequences that were disordered at low concentrations have recently been shown to form quaternary structures through self-assembling helical building blocks.…”

Multivalent foldamer-based affinity assay for selective recognition of Aβ oligomers

“…Somatostatin receptor [7] GLP-1 receptor, [8] PTHR1, [9] the p53-hDM2, [10] the BH3-Bcl-x L [11,12] and the VEGF-VEGFR1 [13] interactions, the gp41 virus cell infusion protein assembly [14], the γ-secretase enzyme [15] and amyloid aggregation. [16] Whilst biomimetic molecular recognition can be achieved with foldamers, their application in therapy, diagnostics and as analytical tools is still a major challenge.…”