Stapled β-Hairpins Featuring 4-Mercaptoproline

Abstract

Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel β-strands, and then we incorporated that staple within a β-hairpin peptide. The hairpin uses 4-mercaptoproline as a novel staple component, which contributes to a unique, kinked structure. The stapled hairpins show a high degree of structure in aqueous solution, excellent resistance to degradation in cell lysates, and cytosolic penetration at micromolar concentrations. They also overlay with a unique subset of kinked hairpin motifs at protein− protein interaction interfaces. Thus, these scaffolds represent promising starting points for developing inhibitors of cellular protein−protein interactions.

Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy

“…Based on the dihedral angles of bound Pen3- ortho and Ramachandran plots of proline and pre-proline residues, , we hypothesized that a 4-mercaptoproline (4MP) residue within the staple would favor Pen3- ortho ’s GABARAP-selective binding conformation. 4MP conformationally restricts both the backbone and the staple, and we recently reported its use as a valuable building block for peptide stapling for non-α-helical peptides . Testing a panel of Pen3- ortho analogues with 4MP in the third position (parent peptide MP3), we found that 4MP substitution maintained high GABARAP affinity while decreasing binding affinity to LC3B for all staple geometries (Figure a).…”

“…Covalent linking of side chains, or “peptide stapling,” is a promising strategy to minimize these limitations . While most stapled peptides are α-helical, we and others have shown that appropriately designed staples can improve the properties of peptides in other conformations as well. ,− Based on the crystal structure of K1 bound to GABARAP, we designed peptides Cys5, Cys4, and Cys3, which stapled K1 in ( i , i + 3), ( i , i + 4), and ( i , i + 5) positions, respectively (Figure a). We stapled each peptide using dithiol bis-alkylation with ortho- , meta- , and para- dimethylbenzene linkers, allowing for a “diversity-oriented stapling” approach, where the staple geometry was varied (Figure a). ,, Stapling in ( i , i + 3) positions (the Cys5 peptides) reduced binding to both GABARAP and LC3B compared to K1.…”

“…Proteolytic degradation is a liability for peptides, but artificial amino acids and staples can provide resistance. We measured the stability of selected GABARAP ligands following 1, 3, and 5 h incubation in freshly prepared HeLa cell lysate, a high-stringency assay for measuring susceptibility to biological degradation. ,, K1 was degraded rapidly, with only 9% remaining after 1 h. Incorporating artificial amino acids such as tert -butyl alanine conferred some protease resistance, with 34% remaining at 1 h and 4% at 5 h (Figure S28). Stapling conferred even more resistance.…”

“…4MP conformationally restricts both the backbone and the staple, and we recently reported its use as a valuable building block for peptide stapling for non-αhelical peptides. 32 Testing a panel of Pen3-ortho analogues with 4MP in the third position (parent peptide MP3), we found that 4MP substitution maintained high GABARAP affinity while decreasing binding affinity to LC3B for all staple geometries (Figure 4a). These data, along with the observation S1).…”

A Recombinant Approach For Stapled Peptide Discovery Yields Inhibitors of the RAD51 Recombinase

“…[9], [10] Recently, alternative structural elements, such as βhairpins and loops have been successfully utilised for stapling. [11], [12] Stapling of the nucleophilic side chain of cysteines has been explored as an attractive alternative to RCM, as it avoids the use of non-natural amino acids and metal catalysts. [13] Stapling of cysteines can be done in mild, biocompatible conditions, allowing it to be used in the context of affinity selections of combinatorial libraries.…”