Advancing Peptide Synthesis Through Stapled Peptides

August 2, 2013
Patricia Van Arnum
Pharmaceutical Technology

Volume 37, Issue 8

Stapled peptides offer promise to enable cell permeability, binding to therapeutic targets, and modulation of biological pathways.

Advancing Peptide Synthesis Through Stapled Peptides (SHUNYU FAN/GETTY IMAGES) As a drug type, peptides offer certain benefits, such as specificity and potency, but they also present challenges, such as poor stability and short half-life. Stapled peptides, small modified helical proteins, are an emerging class of peptides that seek to address these limitations. These alpha-helical peptides have structural and functional properties that enable them to penetrate into the cell, bind to the therapeutic target, and modulate biological pathways.

Peptides as drugs

Patricia Van Arnum Although peptides have the size and functionality to effectively modulate intracellular protein–protein interactions, they often do not permeate cells and, therefore, are used to modulate extracellular targets such as receptors (1–3). The majority of peptide candidates target extracellular molecules with less than 10% binding to intracellular targets, according to an analysis of the peptide drug pipeline by the Peptide Therapeutics Foundation (1, 4). The most common extracellular targets were G-protein coupled receptors (GPCRs), which include nearly 1000 transmembrane proteins that activate cellular response. During 2000–2008, 60% of peptides entering clinical development targeted GPCRs, and most had agonist activity. Although peptides represent a small portion of total drug candidates, the number of peptide drugs entering clinical development has increased during the past several decades, according to the Peptide Therapeutics Foundation analysis, which excluded insulins (1, 4). The study found that the average number of new peptide candidates entering clinical development in the 1970s was 1.2 per year, which rose to 4.6 per year in the 1980s, 9.7 per year in the 1990s, and 16.8 per year through 2000–2008 (1, 4).

On a commercial level, there are several peptide drugs that have reached blockbuster status. These include: Teva Pharmaceutical's Copaxone (glatiramer acetate), an L-glutamic acid polymer with L-alanine, L-lysine and L-tyrosine; AbbVie's Lupron (leuprolide acetate), a synthetic nonapeptide analog of the naturally occurring gonadotropin-releasing hormone (GnRH or luteinising hormone-releasing hormone [LHRH]); AstraZeneca's Zoladex (goserelin acetate), a decapeptide and GnRH agonist and synthetic analog of a naturally occurring LHRH; Novartis' Sandostatin (octreotide acetate), a cyclic octapeptide with pharmacologic actions mimicking those of the natural hormone somatostatin; and Eli Lilly's Byetta (exenatide), a 39-amino-acid peptide amide (1, 4).

Stapled peptides as a solution

Stapled peptides are a nascent class of peptides that use stabilization technology to enhance potency and cell permeability to address pharmacological limitations of small molecules and existing biologics in intracellular protein–protein interactions. Although small molecules are able to penetrate cells, the large binding surfaces for intracellular protein–protein interactions often make small-molecule modulators ineffective. Peptides and proteins have the size and functionality to effectively modulate intracellular protein–protein interactions, but do not permeate cells and, therefore, re used to modulate extracellular targets (1, 2, 5). Stapled peptides seek to resolve those problems. Because many undruggable therapeutic targets include those protein–protein interactions in which alpha-helices are required in lock-and-key-type mechanisms, an approach is to design alpha-helical peptides that have structural and functional properties that enable them to penetrate into the cell, bind to the therapeutic target, and modulate the biological pathway (1, 2, 5).

A commercial path for stapled peptides

Aileron Therapeutics is one company specializing in developing stapled peptides. Its technology stabilizes peptides by "stapling" them with hydrocarbon bonds into an alpha-helix. Once constrained in the alpha-helix structure, the peptides are protected from degradation by proteases. The stabilized alpha-helical peptides can penetrate cells by energy-dependent active transport and typically have a higher affinity to large protein surfaces (1–3, 5).

Aileron was cofounded in 2005 by Gregory L. Verdine, recently named CEO of the genomics company Warp Drive Bio. Verfine, who served as professor of chemistry at Harvard University, director of the Harvard/Dana–Farber Programme in Cancer Chemical Biology, and executive director of the Chemical Biology Initiative at the Dana–Farber Cancer Institute, is noted for advancing the field of stapled peptides. In 2006, Aileron acquired exclusive rights from Harvard University in Massachusetts and the Dana–Farber Cancer Institute to develop and commercialize a drug-discovery pipeline of stapled peptides. In 2006–2007, Aileron licensed rights from the fine-chemicals and technology firm Materia for catalysts used in olefin metathesis. Materia holds the rights to the olefin metathesis technology developed by Robert H. Grubbs, professor at the California Institute of Technology, who was awarded the Nobel Prize in Chemistry in 2005 with Richard R. Schrock and Yves Chauvin for their work in olefin metathesis using ruthenium-based catalysts. Part of the reaction scope of olefin metathesis is ring-closing metathesis (RCM), which transforms a diene into a cyclic alkene and is used to create macrocycles, including bioactive cyclic peptidomimetics. Grubbs was one of the first to offer research describing RCM to tether residues of helical peptides (1, 5).

Aileron is partnered with Roche for stapled peptides. The companies formed a potential $1.1-billion drug-development collaboration in 2010 for the discovery, development, and commercialization of stapled-peptide drugs and later expanded the collaboration. The initial program encompasses up to five programs with the initial two programs targeting oncology, and the third program, launched in late 2011, involving inflammatory diseases. Aileron also is partnered with Novartis and Eli Lilly through the respective venture funds of those companies (1, 5). In May 2013, Aileron announced the completion of the first-in-human study of its lead stapled peptide drug, ALRN-5281, a proprietary, long-acting growth-hormone-releasing hormone agonist for treating orphan endocrine disorders.

Other companies are involved in stapled peptides. In November 2012, MorphoSys, a company specializing in antibody technology, partnered with the Dutch biopharmaceutical company Lanthio Pharma, which is involved with discovering and developing lantipeptides, a class of stapled peptides with high target selectivity and improved drug-like properties, which the company produces through its proprietary technology LanthioPep. The technology is used to identify peptides that are selective for a specific disease target and to stabilize them in their optimal structural conformation for receptor binding. LanthioPep is a Lactococcus lactis based lanthionine-peptide technology and is used to discover peptide therapeutics with increased resistance to peptidase degradation, high receptor specificity, and increased intrinsic activity.

Lanthio Pharma has generated stable, peptidase-resistant lanthionine peptides with specific agonistic activity for a number of GPCR targets, which is a focus area of the company. Many peptide ligands are thought to bind to their GPCR receptors through a "turn motif," which can be stabilized in Lanthio Pharma's peptides with a strong thioether bond. Fixing the turn motif in its optimal receptor binding conformation can result in specific agonistic receptor activation, according to the company. The technology also includes a proprietary bacterial display library capability, which allows for the construction of focused or randomized libraries of lanthionine-peptides. These libraries allow for functional screening and production of peptides for further in vivo and in vitro testing. Therapeutic plasma levels of lantipeptides can potentially be achieved by oral, pulmonary, or subcutaneous delivery. Therapeutic products in Lanthio Pharma's pipeline include a lanthionine-stabilized specific agonist of the AT2 receptor, which has potential in diseases where tissue protection is important, such as fibrosis.

Under the agreement, MorphoSys and Lanthio Pharma will jointly apply their respective technologies to establish lantipeptide-based libraries. MorphoSys received preferred rights to exclusively license the LanthioPep technology for drug discovery and made an equity investment for a minority stake in Lanthio Pharma. Lanthio Pharma also is partnered with US-based Tarix Pharmaceuticals for Lanthio's lead compound PanCyte, a lanthionine-stabilized angiotensin-(1-7) agonistic peptide for treating pulmonary indications. The start of clinical development of PanCyte is expected this year, according to company information.

Stapled peptides advance

Scientists are advancing research in stapled peptides in both drug design and peptide synthesis. Researchers at the New York Structural Biology Center reported on high-resolution nuclear magnetic resonance techniques with dynamic light-scattering to characterize a family of hydrocarbon-stapled peptides with known inhibitory activity against the HIV-1 capsid assembly to evaluate the various factors that modulate activity (1, 6). The researchers reported that helical peptides share a common binding motif but differ in charge, the length and position of the staple. The research showed that the peptides share a propensity to self-associate into organised polymeric structures mediated predominantly by hydrophobic interactions between the olefinic chain and the aromatic side-chains from the peptide. The researchers also detailed the structural significance of the length and position of the staple and of the olefinic bond isomerization in stabilizing the helical conformation of the peptides as potential factors influencing polymerisation (1, 6).

Researchers at the Dana–Farber Cancer Institute, Children's Hospital in Boston, and Harvard University reported the use of hydrocarbon double-stapling to remedy the proteolytic instability of a lengthy peptide (5, 7). Specifically, the researchers applied the stapled approach to Fuzeon (enfuvirtide), a 36-amino-acid peptide that inhibits human immunodeficiency virus Type 1 (HIV-1) infection by targeting the viral fusion apparatus (5, 7).

The researchers noted that enfuvirtide is used as a salvage treatment option because of poor in vivo stability and poor oral bioavailability. To address the proteolytic shortcomings of long peptides as therapeutics, the researchers studied the biophysical, biological and pharmacological impact of inserting all-hydrocarbon staples into the drug (5, 7). The researchers found that the peptide double-stapling created protease resistance and improved pharmacokinetic properties, including oral absorption. The hydrocarbon staples created a "proteolytic shield" by reinforcing the overall alpha-helical structure, which slowed the kinetics of proteolysis and also created a complete blockade of peptide cleavage at the constrained sites in the immediate vicinity of the staple (5, 7). The researchers noted the potential of double-stapling to other lengthy peptide-based drugs.

But for all their promise, some researchers point to limited benefits of stapled peptides. Earlier this year, researchers from the Walter and Eliza Hall Institute of Medical Research in Australia, the University of Melbourne and Roche's Genentech reported on a study involving stapled peptides, specifically for stabilized BimBH3 peptides (BimSAHB), which had reduced affinity for their targets, the pro-survival Bcl-2 proteins (8, 9). The researchers attributed the loss in affinity to disruption of a network of stabilizing intramolecular interactions present in the bound state of the native peptide. They suggested that altering the network may compromise binding affinity, as in the case of the BimBH3 stapled peptide in their study. They also said that cells exposed to these peptides do not readily undergo apoptosis, which indicates that BimSAHB is not inherently cell permeable (8, 9).

Patricia Van Arnum is a executive editor of Pharmaceutical Technology, 485 Route One South, Bldg F, First Floor, Iselin, NJ 08830 tel. 732.346.3072,


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