Structure-based design of synthetic inhibitors of protein-protein interactions requires adept molecular design and synthesis strategies as well as knowledge of targetable complexes. To address the significant gap between the elegant design of helix mimetics and their sporadic use in biology, we analyzed the full set of helical protein interfaces in the Protein Data Bank to obtain a snapshot of how helices that are critical for complex formation interact with the partner proteins. The results of this study are expected to guide systematic design of synthetic inhibitors of protein-protein interactions. We have experimentally evaluated new classes of protein complexes that emerged from this dataset – highlighting the significance of the results described herein.

Summary of recent advancesHerein we review contemporary synthetic and protein design strategies to stabilize the α-helical motif in short peptides and miniature proteins. Advances in organometallic catalyst design, specifically for the olefin metathesis reaction, enable the use of hydrocarbon bridges to either crosslink side chains of specific residues or mimic intramolecular hydrogen bonds with carbon-carbon bonds. The resulting hydrocarbon-stapled and hydrogen bond surrogate α-helices provide unique synthetic ligands for targeting biomolecules. In the protein design realm, several classes of miniature proteins that display stable helical domains have been engineered and manipulated with powerful in vitro selection technologies to yield libraries of sequences that retain their helical folds. Rational re-design of these scaffolds provide distinctive reagents for the modulation of protein-protein interactions.

Mimics of α-helices on protein surfaces have emerged as powerful reagents for antagonizing protein-protein interactions, which are difficult to target with small molecules. Herein we describe the design of a cell-permeable synthetic α-helix based on the guanine nucleotide exchange factor Sos that interferes with Ras-Sos interaction and downregulates Ras signaling in response to receptor tyrosine kinase activation.

Alpha-helices constitute the largest class of protein secondary structures and play a major role in mediating protein-protein interactions. Development of stable mimics of short alpha-helices would be invaluable for inhibition of protein-protein interactions. This Account describes our efforts in developing a general approach for constraining short peptides in alpha-helical conformations by a main-chain hydrogen bond surrogate (HBS) strategy. The HBS alpha-helices feature a carbon-carbon bond derived from a ring-closing metathesis reaction in place of an N-terminal intramolecular hydrogen bond between the peptide i and i + 4 residues. Our approach is centered on the helix-coil transition theory in peptides, which suggests that the energetically demanding organization of three consecutive amino acids into the helical orientation inherently limits the stability of short alpha-helices. The HBS method affords preorganized alpha-turns to overcome this intrinsic nucleation barrier and initiate helix formation. The HBS approach is an attractive strategy for generation of ligands for protein receptors because placement of the cross-link on the inside of the helix does not block solvent-exposed molecular recognition surfaces of the molecule. Our metathesis-based synthetic strategy utilizes standard Fmoc solid phase peptide synthesis methodology, resins, and reagents and provides HBS helices in sufficient amounts for subsequent biophysical and biological analyses. Extensive conformational analysis of HBS alpha-helices with 2D NMR, circular dichroism spectroscopies and X-ray crystallography confirms the alpha-helical structure in these compounds. The crystal structure indicates that all i and i + 4 C=O and NH hydrogen-bonding partners fall within distances and angles expected for a fully hydrogen-bonded alpha-helix. The backbone conformation of HBS alpha-helix in the crystal structure superimposes with an rms difference of 0.75 A onto the backbone conformation of a model alpha-helix. Significantly, the backbone torsion angles for the HBS helix residues fall within the range expected for a canonical alpha-helix. Thermal and chemical denaturation studies suggest that the HBS approach provides exceptionally stable alpha-helices from a variety of short sequences, which retain their helical conformation in aqueous buffers at exceptionally high temperatures. The high degree of thermal stability observed for HBS helices is consistent with the theoretical predictions for a nucleated helix. The HBS approach was devised to afford internally constrained helices so that the molecular recognition surface of the helix and its protein binding properties are not compromised by the constraining moiety. Notably, our preliminary studies illustrate that HBS helices can target their expected protein receptors with high affinity.

Herein we describe a strategy for the preparation of artificial alpha-helices involving replacement of one of the main-chain hydrogen bonds with a covalent linkage. To mimic the C=O...H-N hydrogen bond as closely as possible, we envisioned a covalent bond of the type C=X-Y-N, where X and Y are two carbon atoms connected through an olefin metathesis reaction. Our results demonstrate that the replacement of a hydrogen bond between the i and i + 4 residues at the N-terminus of a short peptide with a carbon-carbon bond results in a highly stable constrained alpha-helix at physiological conditions as indicated by CD and NMR spectroscopies. The advantage of this strategy is that it allows access to short alpha-helices with strict preservation of molecular recognition surfaces required for biomolecular interactions.