Hydrocarbon-stapled peptides: principles, practice, and progress

Abstract

Protein structure underlies essential biological processes and provides a blueprint for molecular mimicry that drives drug discovery. Although small molecules represent the lion's share of agents that target proteins for therapeutic benefit, there remains no substitute for the natural properties of proteins and their peptide subunits in the majority of biological contexts. The peptide α-helix represents a common structural motif that mediates communication between signaling proteins. Because peptides can lose their shape when taken out of context, developing chemical interventions to stabilize their bioactive structure remains an active area of research. The all-hydrocarbon staple has emerged as one such solution, conferring α-helical structure, protease resistance, cellular penetrance, and biological activity upon successful incorporation of a series of design and application principles. Here, we describe our more than decade-long experience in developing stapled peptides as biomedical research tools and prototype therapeutics, highlighting lessons learned, pitfalls to avoid, and keys to success.

Figure 1

Application of ruthenium-catalyzed olefin metathesis to install macrocyclic cross-links into synthetic peptides. Blackwell and Grubbs performed the metathesis reaction on a pair of O-allylserine residues (top), whereas Schafmeister and Verdine employed α,α-disubstituted non-natural amino acids bearing all-hydrocarbon tethers (bottom). The latter approach yielded peptide constructs with marked α-helical stabilization.

Figure 2

Enhanced α-helicity of all-hydrocarbon stapled peptides. Circular dichroism analyses of a series of BH3 peptides demonstrate that stapling can convert unfolded BID (AA 81–101) (A), BAD (aa 103–127) (B), BIM (aa 146–166) (C), and MCL-1 (208–226) (D) peptides (14–20% α-helical content) into α-helices (71–91% α-helical content) in solution (e.g., aqueous potassium phosphate, pH 7).

Figure 3

Building blocks of all-hydrocarbon peptide stapling. (A) A series of chiral non-natural amino acids are inserted at i, i + 4 or i, i + 7 positions and the terminal olefins cross-linked by RCM, yielding cross-links that span one or two helical turns, respectively. For example, S5–S5 pairs have been substituted at i, i + 4 positions, and S8–R5 or S5–R8 pairs have been substituted at i, i + 7 positions to generate single- or double-stapled peptides. (B) Two synthetic approaches that we have used to generate the stapling amino acids employ the oxazinone or BPB-Ni(II)-Ala chiral auxiliaries to enforce the desired stereochemistry.

Figure 4

Generating a library of stabilized α-helices by staple scanning. Ideally, the structure of a helix-in-groove interaction can help guide the selection of staple insertion points to maximize α-helical stabilization while avoiding interference with critical, native contact points between the helix and groove. Sequential placement of staples along the entire length of the peptide sequence yields a library of constructs for structure–activity relationship analyses. We have used this staple scanning approach to identify optimal staple positions for structural stabilization, elucidate key residues and contact surfaces for ligand–target interaction, and generate negative control constructs for biological studies.

Figure 5

Design and derivatization of stapled peptides for a diversity of research applications. We have generated stapled peptides for (1) cellular studies by optimizing α-helicity and adjusting overall charge to the 0 to +2 range, (2) PRE NMR analyses by optimizing solubility with overall negative charge and appending differentially localized spin labels, (3) fluorescence polarization binding and cellular uptake analyses by N-terminal derivatization with FITC, (4) in vivo PK and extracellular targeting studies of lengthy α-helices by inserting two staples, (5) protein interaction discovery and helix binding site identification by inserting photoreactive non-natural amino acids along the length of an N-terminally biotinylated stapled peptide followed by affinity capture and mass spectrometry analysis.

Figure 6

Structural analysis of stapled peptide helices. (A, B) Examination of a series of differentially stapled MCL-1 BH3 (aa 208–226) (A) and p53 transactivation domain (aa 14–29) (B) peptides by circular dichroism demonstrates the importance of staple position in optimizing α-helical stabilization. Whereas the majority of MCL-1 SAHB constructs manifest substantial structural stabilization compared to the unmodified MCL-1 BH3 peptide (A), only one of the sampled positions in the p53 sequence yielded a peptide with marked α-helicity. These data demonstrate that installing a hydrocarbon staple at any one location does not guarantee structural enhancement, but sampling a series of positions can typically yield a construct or a panel of constructs with the desired properties. (C, D) X-ray structures of the stapled peptide/target protein complexes MCL-1 SAHB/MCL-1 (C) and SAH-p53-8/HDM2 (D) demonstrate the reinforced α-helical structure of the peptide ligands and the capacity of the staple itself to engage the protein surface, resulting in enhancement of binding activity without compromising specificity.

Figure 7

Protease resistance of hydrocarbon-stapled peptides. (A, B) A mechanistic analysis of peptide fortification by hydrocarbon stapling revealed that the average α-helicity in solution of a doubly stapled lengthy peptide SAH-gp41(A,B) vs the corresponding tetrasubstituted but unstapled analogue UAH-gp41(A,B) was the same (A), yet the proteolytic half-life of the doubly stapled construct was prolonged by 24-fold compared to the unmodified peptide, whereas the tetrasubstituted but unstapled analogue showed only a 3-fold difference (B). (C) Proteomic analysis of the digestion products revealed that peptide double stapling slowed the kinetics of proteolysis at sites distal to the staple and completely prevented hydrolysis at sites flanked by or immediately adjacent to the staple. Notably, the tetrasubstituted but unstapled analogue was unable to achieve this degree of protection. (D) The dramatic antiproteolysis effect of hydrocarbon double stapling was reflected by a 192-fold enhancement in half-life of SAH-gp41(A,B) compared to the corresponding unmodified peptide in the presence of pepsin at pH 2. (E) The striking in vitro proteolytic stability of SAH-gp41(A,B) at acidic pH prompted us to explore its oral bioavailability after administration to mice by oral gavage. SAH-gp41(A,B) achieved measurable and dose-dependent plasma concentrations, in fully intact form, within 30 min of oral administration, whereas the corresponding unmodified construct was not detectable (ND).

Figure 8

Deploying stapled peptides for biological investigation. Workflows for using stapled peptides in (A) in vitro biochemical, structural, and functional studies and (B) cellular and in vivo analyses.

Figure 9

Stapling down the facts on BIM SAHBs. In order to accomplish a challenging NMR analysis of the hit-and-run interaction between BIM BH3 and BAX (left), we adjusted the sequence of our prototype high α-helicity, high affinity, and cell permeable BIM SAHB_A (146–166) peptide^, (right) to enhance its solubility and weaken (i.e., slow down) its BAX-activating capability (left). Czabotar and colleagues from WEHI and Genentech also successfully applied this refashioned BIM SAHB_A (145–164) peptide for structural determination (left) (“Yes” arrow). However, the authors misapplied the weakened-by-design BIM SAHB_A (145–164) construct in binding and cellular studies (“No” arrow) and predictably observed no cellular activity, yet broadly concluded that stapling BIM BH3 does not enhance its binding affinity or biological activity. In response to our recent correspondence, Czabotar and co-authors have now tested the correct BIM peptide (right) in leukemia cells and successfully reproduced our published results.To facilitate the successful application of peptide stapling, a rigorous and detail-oriented approach is required and includes careful consideration of the sequence, biophysical, biochemical, structural, cellular uptake, and biological properties of discrete stapled peptide constructs (“Yes” arrows).

Figure 10

Growth of stapled peptide applications for biomedical discovery and drug development. (A) Since the original reports of the all-hydrocarbon cross-linked peptide helix and its proof-of-concept utility for signal transduction research, cellular delivery, and therapeutic targeting, there has been increased accessibility to and successful application of the technology by us and many other laboratories, as indicated by the growing number of yearly stapled peptide publications. (B) Stapled peptides serve as versatile probes for protein interaction research and as prototype therapeutics for modulating extracellular and intracellular protein targets.