Stapled Peptides

Peptide mimics useful for the modulation of protein and receptor signaling and subsequent gene expression

By Klaus D. Linse

Synthetic peptides that specifically bind to proteins such as cytokines, protein hormones, and nuclear hormone receptors offer an alternative approach to small molecules for the modulation of protein and receptor signaling and subsequent gene expression. LaBelle et al (2012) published a research article recently that showed that a stabled helical BCL-2-interacting mediator of cell death (BIM) peptide overcomes apoptotic resistance in hematologic cancers.


Figure 1: Sequences of BIM SAHBA and R153D mutant and structure of the stabled BIM BH3 helix peptide (Source: LaBelle et al., 2012).

A hydrocarbon-stabled peptide modeled after the BIM BH3 helix broadly targeted BCL-2 family proteins with high affinity (Figure 1). This peptide blocked inhibitory anti-apoptotic interactions thereby directly triggering proapoptotic activity inducing dose-responsive and BH3 sequence-specific cell death of hematologic cancer cells. The therapeutic potential of the peptide was established by the selective activation of cell death in the aberrant lymphoid infiltrates of mice reconstituted with BIM-deficient bone marrow and in a human AML xenograft model. These results indicate that the BCL-2 family interaction network that lies at the crossroads of the cell’s life-death decisions can be targeted with therapeutic compounds that selectively modulate the network.  Apoptosis regulator Bcl-2 homology (BH) domain is a family of evolutionarily related proteins that govern mitochondrial outer membrane permeabilization (MOMP) and can be either pro-apoptotic or anti-apoptotic. There are a total of 25 genes in the Bcl-2 family known to date.

The inactivation of apoptosis is believed to be the main reason leading to the development of cancer. Response to cancer therapy is also governed by apoptotic responses. In some cases it has been observed that apoptosis is not the main mechanism for the death of cancer cells in response to a treatment, thus leading to resistance to the treatment. Apoptosis is a highly regulated, energy-dependent cellular program, whereby the cell activates a signaling cascade that leads to cell death without triggering an inflammatory response mediated by two distinct pathways – extrinsic and intrinsic cell death pathways.

The extrinsic pathway is activated when death ligands, such as Fas ligand or tumor necrosis factor (TNF), bind to their cognate receptors at the plasma membrane. This causes homo-trimerization of the receptor and recruitment of specific adaptor proteins, such as Fas-associated death domain and procaspase-8, into a death-inducing signaling complex. This, in turn, leads to activation of initiator caspase-8, which subsequently activates effector caspases. In case of the intrinsic pathway, the mitochondria play a central role in the integration and execution of a wide variety of apoptotic signals, including loss of growth factors, hypoxia, oxidative stress, and DNA damage. The mitochondria provide the energy required for execution of the apoptotic program and the release of proapoptotic proteins such as cytochrome c, endonuclease G, and apoptosis-inducing factor. The release of cytochrome c leads to apoptotic protease-activating factor (Apaf-1)-mediated activation of initiator caspase-9, which in turn activates effector caspases. Thus the extrinsic and intrinsic pathways have different initiator caspases but converge at the level of the effector caspases.

The intrinsic pathway of apoptosis is regulated by members of the Bcl-2 family as illustrated in figure 2. The Bcl-2 family is composed of pro- and anti-apoptotic proteins that share up to four conserved regions known as Bcl-2 homology (BH) domains. Anti-apoptotic members such as Bcl-2 and Bcl-XL contain all four subtypes of BH domains and promote cell survival by inhibiting the function of the pro-apoptotic Bcl-2 proteins. Anti-apoptotic Bcl-2 proteins have been reported to protect cells from many different apoptotic stimuli and are important for cell survival. In some circumstances, Bcl-2 and Bcl-XL are targets of caspases, and cleavage of these proteins converts them from pro-survival to pro-apoptotic molecules that are able to induce cytochrome c release from the mitochondria.

Bcl 2 proteins

Figure 2:  Domain structure of Bcl-2 family proteins. Bcl-2 homology (BH) and transmembrane (TM) domains are indicated.

BH3 is a spring-like shaped α-helix with amino acids on its surface that bind to and inhibit anti-death proteins such as BCL-2, as well as activating pro-death proteins under certain circumstances. However, when the BH3 peptide is synthesized its shape is lost and its functionality impaired. To stabilize the peptide, Loren D Walensky and colleagues at the Dana-Farber Cancer Institute used a chemical strategy called the hydrocarbon-stapling technique developed by Verdine and Helinski. Some amino acids in the natural sequence are replaced with synthetic ones bearing hydrocarbon ‘tethers’. The tethers link to form chemical ‘staples’, which reinforce the α-helical structure. The new peptide retains its biological activity and actually binds more strongly to the BCL-2 target, the researchers report. The stapled peptides, called “stabilized alpha-helix of BCL-2 domains” (SAHBs), were helical, protease-resistant, and cell-permeable molecules that bound with increased affinity to multidomain BCL-2 member pockets. Hydrocarbon stapling generated BH3 peptides with improved pharmacologic properties. Different BCL-2 proteins are implicated in different cancers, but all contain α-helical BH3 domains.

Stapled peptides may offer new ways to treat so called “undruggable” diseases. Available drugs, primarily small molecules and therapeutic proteins, address only an estimated 10 to 20 % of all identified therapeutic targets within the human body.

What makes stabled peptides different?

Proteases, naturally present in the human body, can only recognize and digest peptides when they are unfolded. Therefore, if the peptides are locked into certain folded shapes, they are protected from the attack of the proteases and will longer remain in the tissues where the targeted compounds reside. In the past, many attempts have been made to stabilize peptides by chemical modifications, however, often some if not all of their biological activity is lost or the modified peptides cannot penetrate cells.

Key steps for the design of stabling peptides involves using a cross-linking chemistry that locks the peptides into a a-helical shape that mimics the structure found at the interface of many protein-protein interactions. One prominent method is the hydrocarbon stapling approach.

What makes the peptide cell permeable?

The following researchers think they have the answer. Muppidi et al., (2012) showed that the direct chemical modifications of helical peptides have provided a simple and effective means to ‘translate’ bioactive helical peptides into potential therapeutics targeting intracellular protein-protein interactions. The researchers have shown that the distance-matching bisaryl cross-linkers can reinforce peptide helices containing two cysteines at the i.i+7 positions and confer cell permeability to the cross-linked peptides. The group reported the first crystal structure of a biphenyl cross-linked Noxa peptide in complex with its target Mcl-1 at a 2.0 Å resolution. Guided by the structure, they remodeled the surface of the cross-linked peptide through side chain substitution and N-methylation, and obtained a pair of cross-linked peptides with substantially increased helicity, cell permeability, proteolytic stability, and cell-killing activity in Mcl-1-overexpressing U937 cells.

Dimartino’s group described the solid-phase synthesis of hydrogen-bond surrogate-derived artificial a-helices by a ring-closing metathesis reaction. The researchers found that the Hoveyda-Grubbs catalyst gave high yields of the macrocycle irrespective of the peptide sequence. Furthermore, they concluded that hydrogen-bond surrogate derived artificial a-helices can be efficiently synthesized on solid phases with this catalyst and that the synthesis of the a-helices does not require the preparation of new enantiomerically pure amino acids and can be achieved by standard peptide synthesis protocols with easily available monomers. The number of researchers that now report on the synthesis of these peptides has increased considerably since Blackwell, and Grubbs in 1998 reported the synthesis of the first generation of stapled peptides that ranged in size from 12 to 35 amino acids in length.

Harrison et al. published a paper in 2010 that reported the downsizing of proteins to short water-stable a-helices that maintain biological potency. The much shorter cyclic peptides mimic the a-helical parts of the protein structure and appear to be more stable than the parent proteins and have biological activity as well.

cPentapeptides GPCR ligand nociceptin

Figure 3: (Left) α-helices composed of cyclic pentapeptides. Different distributions of variable side chains (gray spheres) and linking bridges (green) allow the design of different helix mimics for binding to different receptors. (Right) GPCR-ligand nociceptin. The nociceptin peptide with residues 1 to 17 is a super agonist of ORL-1. ORL 1 or Opioid Like Receptor 1 is a member of the family of rhodopsin-like G protein-coupled receptors (GPCR) that shares functional and structural homology with Opioid Receptors systems and is involved in a variety of biomedical important processes, such as anxiety, nociception, feeding, and memory. The mimic of this peptide is shown below the sequences (Harrison et al. 2010).


The building block is a small cyclic pentapeptide (KAAAD), which structure is similar to a lactam between residues 1 and 5. The joining of two or more of these peptides allows the design of stabled peptides with more turns, whereas the replacement of alanines allows for the synthesis of more possible mimics of the endogenous protein. The researchers showed that an analog of nociceptin is a potent agonist at ORL-1 (see figure 2). Furthermore, the constrained peptides are stable in human serum for over 24 hours, as compared to their uncyclized  peptides, which are degraded rapidly.

Verdine and Hilinski in 2010 report on the design and synthesis of a new class of synthetic miniproteins locked into their bioactive α-helical fold through the site-specific introduction of a chemical brace, an all-hydrocarbon stable in ‘Methods of Enzymology.’ The researchers discuss considerations crucial to the successful design and evaluation of potent stapled peptide interactions to facilitate the broad application of this technology to intractable targets of both basic biologic interest and potential therapeutic value. The synthesis of three types of all-hydrocarbon stapled peptides is described (See figure 4). The synthesis of stapled peptides is usually performed with standard Fmoc-chemistry based solid phase synthesis with modifications to allow for the cyclization reaction by metathesis prior to labeling or tagging the peptide with fluorophores, biotin or other functional groups (See Verdine and Hilinski, 2012, for more details).

crosslinking aa NOTCH CSL

Figure 4: (Left) α-Methyl, α-alkenylglycine cross-linking amino acids are incorporated during solid-phase peptide synthesis. The schematics for the synthesis of an i, i+3 stapled, an i, i+4 stapled, and an i, i+7 stapled peptide is illustrated . (Right)  The 3D model of a stapled peptide targeting the NOTCH/CSL binary complex is shown. A kinked α-helix from the MAML transcriptional coactivator protein binds to a cleft created by the NOTCH/CSL complex. The region of MAML from which the bioactive SAHM1 stapled peptide was designed is indicated with a box. MAML residues E28 and R32 were replaced with S5 cross-linking amino acids to form SAHM1. PDB: 2F8X. (Source: Verdine and Hilinski, 2012).

Extensive biophysical characterization can be done with a diverse portifolio of techniques such as Circular Dichroism (CD), to evaluate the presence and nature of the a-helical structure, Fluorescence polarization (FP), and Surface Plasmon Resonance (SPR), to quantitatively measure the binding constants that describe the interaction of stabled peptides with a recombinant target protein. Furthermore, the presence of the final peptide as well as proteolytic susceptibility can be measured using assays based on HPLC– or Mass Spectrometry (MS)-based detection of intact peptides.  The cell permeability of the peptides can be evaluated using flow cytometry and confocal fluorescence microscopy which require fluorescenctly labeled peptides. The ability of the peptides to interact with the target can be tested in vitro by in vitro immunoprecipitation (IP) and pull-down assays. The final step toward validating the therapeutic usefulness of the finally selected peptide mimetic is a test for in vivo efficacy. A relevant biological model system, such as a tumor progression murine model for cancer, is needed for this test. This is a final proof-of-principle in vivo experiment before clinical trials can be started.

The α-helix motif occurs frequently in biologically important protein interactions, so stapling might allow peptides to be used as drugs in many different applications.Thus hydrocarbon stapling of native peptides may provide a useful strategy for experimental and therapeutic modulation of protein-protein interactions in many signaling pathways.


Helen E. Blackwell, Robert H. Grubbs Highly Efficient Synthesis of Covalently Cross-Linked Peptide Helices by Ring-Closing Metathesis; Angewandte Chemie International Edition Volume 37, Issue 23, pages 3281–3284, December 17, 1998.

Gianluca Dimartino, Deyun Wang, Ross N. Chapman, and Paramjit S. Arora;   Solid-Phase Synthesis of Hydrogen-Bond Surrogate-Derived a-Helices ORGANIC LETTERS 2005 Vol. 7, No. 12 2389-2392

Åsa B. Gustafsson and Roberta A. Gottlieb 2007. Am J Physiol Cell Physiol 292: C45-C51

Rosemary S. Harrison, Nicholas E. Shepherd, Huy N. Hoang, Gloria Ruiz-Gómez, Timothy A. Hill, Russell W. Driver, Vishal S. Desai, Paul R. Young, Giovanni Abbenante, and David P. Fairlie;  Downsizing human, bacterial, and viral proteins to short water-stable alpha helices that maintain biological potency.  Proc Natl Acad Sci U S A. 2010 June 29; 107(26): 11686–11691

James L. LaBelle, Samuel G. Katz, Gregory H. Bird, Evripidis Gavathiotis, Michelle L. Stewart, Chelsea Lawrence, Jill K. Fisher, Marina Godes, Kenneth Pitter, Andrew L. Kung, and Loren D. Walensky; A stapled BIM peptide overcomes apoptotic resistance in hematologic cancers. J Clin Invest. 2012 June 1; 122(6): 2018–2031.

Avinash Muppidi , Kenichiro Doi, Selvakumar Edwardraja, Eric J Drake, Andrew M. Gulick, Hong-Gang Wang , and Qing Lin;    Rational Design of Proteolytically Stable, Cell-Permeable Peptide-Based Selective Mcl-1 Inhibitors  J. Am. Chem. Soc., Just Accepted Manuscript. 2012.

Verdine GL, Hilinski GJ.; Stapled peptides for intracellular drug targets.  Methods Enzymol. 2012; 503:3-33.

Loren D. Walensky et al. 2004. Activation of Apoptosis in Vivo by a Hydrocarbon-Stapled BH3 Helix, Science 3 Vol. 305  No.5689 pp- 1466 – 1470

Lin Zhang, Lihua Ming, and Jian Yu 2007. BH3 mimetics to improve cancer therapy; mechanisms and examples. Drug Resist Updat. 2007 December; 10(6): 207–217.

Categories: Cancer, conjugation, Peptide Mimics, Peptide Synthesis, peptides, Stapled Peptides, Synthesis

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1 reply

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