Stapled Peptides

Hydrocarbon stapled peptides are peptides locked into their bioactive alpha-helical conformation through site-specific introduction of a chemical brace, an all-hydrocarbon staple. The idea of peptide stapling was introduced to overcome the limitations of two broad classes of therapeutic agents (small molecules and protein biologics) in targeting intracellular protein-protein interactions. Small molecules only work on proteins with a specific feature on their surfaces and most protein biologics do not penetrate into cells. Because stapled peptides are locked into a stabilized α-helical structure (the most common element of protein secondary structures), they can easily penetrate cells. As a rapidly emerging class of next-generation drugs, stapled peptides are expected to combine the synthetic manipulability and cell-penetrating ability of small molecules with the three-dimensionality and versatile target recognition ability of biologics. CPC Scientific has extensively developed stapled peptide structures and is the company of choice to manufacture your stapled peptide requirements. Our technical consultants would be happy to discuss your structural design needs with you at any time.

Chemistry

Grubbs catalyst's are routinely used in olefin metathesis to incorporate hydrocarbon staples into peptides.

Two distinct conformational strategies are utilized to induce and stabilize an α-helical structure, namely, α,α-di-substitution (helix nucleation by α-methylation) and macro-cyclic bridge formation (conformational constraint).

Incorporating a staple into a peptide structure entails the integration of two appropriately-spaced, α-methyl, α-alkenylglycine residues, having defined stereochemical configuration and alkene chain length, followed by ruthenium-mediated olefin metathesis before cleavage from the synthesis resin and deprotection, to yield the stapled peptide. Three different types of all hydrocarbon staples are shown in the figure, demonstrating the creation of a stabilized α-helix in a peptide. Approximately one turn of the helix would be i and i+3 (or i, i+4) and two turns of the helix would be i, i+7; three turns of the helix would be i, i+11. For information about the R/S descriptors shown in the figure, please see the Cahn-Ingold-Prelog priority rules for naming stereoisomers.

Stapled Peptides in Drug Design

The introduction of a hydrocarbon staple confers high levels of α-helical content and results in:

• Better target affinity (5 to 5,000-fold increase)
• Increased proteolytic resistance and serum half- life
• Cell penetration through endocytic vesicle trafficking
• Targeting of either extracellular or intracellular proteins
• Disruption of protein-protein interactions
• Non-immunogenicity
• Viable pharmacokinetics and in vivo stability

Novel stapled peptide (prepared by CPC Scientific) bound to the coactivator peptide site of estrogen receptor alpha. Phillips, Chris, et al. Journal of the American Chemical Society 133.25 (2011): 9696-9699.

Phillips, Chris, et al. "Design and structure of stapled peptides binding to estrogen receptors." Journal of the American Chemical Society 133.25 (2011): 9696-9699.

Stapled Peptide Citations

Hydrocarbon-stapled peptide corresponding to the BH3 domain of BIM, BIM SAHBA: N-acetylated 145EIWIAQELRS5IGDS5FNAYYA164-CONH2, S5 = non-natural amino acid inserted for olefin metathesis, was synthesized, purified and characterized as previously described by CPC Scientific (Gavathiotis et al., 2008).

Stapled Peptides by Click Chemistry

The high efficiency and mild conditions of “click” reaction (Copper-catalyzed Huisgen 1,3-dipolar cycloaddition reaction) combined with the ease of synthesis of the necessary unnatural amino acids, allows for facile synthesis of triazole-stapled peptides. For example, a combination of L- Nle (εN3) and D-Pra (D-propargylalanine) substituted at the i and i+4 positions, can be used for the generation of single triazole-stapled peptides.

Modifications of stapled peptides

Stapled peptide modifications typically fall into two categories: a fluorescent label or an affinity tag. Two of the most common moieties appended to the N-terminus of stapled peptides are fluorescein, which can be used for studies of intracellular uptake and biophysical characterization, and biotin, which can be used for biophysical characterization and assessment of in vitro target interaction. It is generally desired to include a flexible molecular spacer to isolate the modification from the core of the stapled peptide.

• N-acetylation
• Linker attachment (β-alanine, mini-PEG, etc.)
• Fluorescent labeling (FITC, 5-FAM, etc.)

Protein Targets

Stapled peptides have been studied in the targeting of several proteins relevant in diseases such as cancer, diabetes, HIV, and atherosclerosis. These proteins include:

• B-cell lymphoma 2 (Bcl-2)
• B-cell lymphoma-extra large (Bcl-xL)
• Bcl-2-associated X protein (Bax)
• Induced myeloid leukemia cell differentiation(Mcl-1)
• Glucokinase (GK)
• Murine double minute 2 (Mdm2)
• Notch/CSL
• HIV-1 capsid and HIV-1 gp41
• ATP-binding cassette transporter (ABCA1)
• Estrogen receptor