The Marriage of Small Molecules and Biologics

May 2, 2011
Patricia Van Arnum
Pharmaceutical Technology

Volume 35, Issue 5

Approaches in using small-molecule and peptide synthesis offer promise in widening the scope of drug candidates.

The ongoing task of drug development is to move promising discovery candidates into commercial production. Different modalities of small-molecule or biologic-based drugs offer relative advantages and disadvantages in achieving these goals. Recent efforts in drug development seek to marry the best of both modalities with specialized approaches, such as stapled peptides and other improvements in peptide synthesis.


Stapled peptides

Stapled peptides use peptide-stabilization technology to enhance potency and cell permeability of a drug. Although the concept of stapled peptides is not new, stapled peptides as a field came into greater prominence last year when Roche signed a drug-development deal worth up to $1.1 billion with the biopharmaceutical company Aileron Therapeutics to discover, develop, and commercialize stapled peptides. Under the agreement, which was announced in August 2010, Roche is guaranteeing at least $25 million in funding for technology-access fees and continued research and development efforts by Aileron. The company is eligible to receive up to $1.1 billion in payments based on discovery, development, regulatory, and commercialization milestones if drug candidates are developed for five undisclosed drug targets in the following areas: oncology, virology, inflammation, metabolism, and central nervous system.

Patricia Van Arnum

Stapled peptides are designed 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. Although peptides and proteins 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). These limitations of small molecules and existing biologics make a vast array of potential drug targets "undruggable." Approximately 80% of potential drug targets are considered "undruggable" by either modality (1, 2).

Peptides face certain limitations as drugs. They lack the ability to enter cells, are inherently unstable within the body, are rapidly broken down into inactive fragments by circulating enzymes, such as proteases, and are quickly filtered from the bloodstream by the kidneys. Stapled peptides seek to resolve those problems. Because many "undruggable" therapeutic targets include those protein–protein interactions in which a-helices are required in lock-and-key-type mechanisms, an approach is to design a-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).

Aileron stabilizes peptides by "stapling" them with hydrocarbon bonds into an a-helix. Once constrained in the a-helix structure, the peptides are protected from degradation by proteases. The stabilized a-helical peptides can penetrate cells by energy-dependent active transport and typically have a higher affinity to large protein surfaces (1, 2).

Aileron was cofounded in 2005 by Gregory L. Verdine, chair of Aileron's scientific advisory board, professor of chemistry at Harvard University, director of the Harvard/Dana–Farber Program in Cancer Chemical Biology, and executive director of the Chemical Biology Initiative at the Dana–Farber Cancer Institute. In 2006, Aileron acquired exclusive rights from Harvard University 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 (3, 4).

In 2008, Aileron acquired exclusive rights from New York University for additional methods to stabilize peptides and peptidomimetics. In 2009, Aileron received $40 million in venture capital funding, which included funding from four pharmaceutical venture-capital funds: SR One (GlaxoSmithKline's venture capital fund), the Novartis Venture Fund, Lilly Ventures (Eli Lilly's venture capital fund), and the Roche Venture Fund.

Verdine recently spoke at the American Chemical Society's (ACS) National Meeting & Exposition in Anaheim, California, in late March 2011, to provide an update of his research at Harvard with respect to stapled peptides. "Our stapled peptides can overcome the shortcomings of drugs of the past and target proteins in the body that were once thought to be undruggable," he said in a Mar. 28, 2011, ACS press release. "They are a genuinely new frontier in medicine."

Verdine highlighted two stapled-peptide drug candidates that respectively target colon cancer and asthma. The colon-cancer stapled peptides inhibit activity of the protein β-catenin, which when present in a hyperactive form, causes cell to grow in an uncontrolled way. This protein has been linked with an increased risk of colon cancer and other types of cancer, including skin, brain, and ovarian cancer. When introduced to human colon cancer cells in laboratory cultures, the stapled peptides reduced the activity of β-catenin by 50%, according to the ACS release.

In a second development, Verdine reported on what he identified to be the first stapled cytokines for treating asthma. Cytokines are hormone-like proteins secreted by the cells of the immune system and other body systems that help orchestrate intercellular signalling. The stapled cytokines moderate the activity of the cytokine, interleukin–13, which asthma patients produce in abnormally large amounts that contribute to asthma attacks, according to the ACS release.

In another development, researchers at the Dana–Farber Cancer Institute, Children's Hospital in Boston, and Harvard University recently reported the use of hydrocarbon double-stapling to remedy the proteolytic instability of a lengthy peptide (5). 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.

Fuzeon is marketed by Roche, which developed the drug with the biopharmaceutical company Trimeris. Roche is responsible for the manufacture, sales, marketing, and distribution of Fuzeon. Roche manufactures bulk quantities of Fuzeon drug substance in its Boulder, Colorado, facility and produces finished drug product from bulk drug substance at other Roche facilities, according to Trimeris' 2010 annual filing with the US Securities and Exchange Commission. The finished drug product is shipped to another Roche facility for distribution. The drug had 2010 sales of $88 million.

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). 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 a-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). The researchers noted the potential of double-stapling to other lengthy peptide-based drugs.

Earlier this year, researchers at the University of Buffalo reported ways of stapling peptide helices. Their approach, dubbed "photoclick stapling," involves the photo-induced 1,3-dipolar cycloaddition reactions (i.e., photoclick chemistry) involving small-ring heterocycles and simple alkenes for both in vitro and live-cell applications. The researchers specifically reported on the photo-induced 1,3-dipolar cycloaddition reaction to staple a peptide dual inhibitor of the p53–Mdm2/Mdmx interactions. The researchers reported that a series of stapled peptide inhibitors were efficiently synthesized and showed dual inhibitory activity in an enzyme-linked immunosorbent assay. The positively charged, stapled peptides showed enhanced cellular uptake along with modest in vivo activity (6). In addition to extending the stapled peptide approached targeting p53–Mdm2/Mdmx interactions, the researchers also are examining BH3–Bcl2/Bcl–xL interactions as potential anticancer therapies.

"There is a lot of potential here." said Qing Lin, assistant professor at the University of Buffalo and lead researcher, in a Feb. 5, 2011, University of Buffalo press release. "Our chemistry is unique. There are not many new drug targets out there today, which partly explains the declining number of FDA-approved new drugs in recent years. So there's a need to come up with new technologies that can overcome this barrier. To this end, stapled peptides could open up a whole host of new targets for therapies."

Other approaches

Improving peptide synthesis also is an area of ongoing research. Researchers from Vanderbilt University recently reported their efforts in overcoming a limitation in peptide synthesis, the incorporation of non-natural amino acids into the peptide chain. The researchers noted that creation of amide bonds typically use methods that principally are based on dehydrative approaches or oxidative and radical-based methods. Generally, carbon and nitrogen bear electrophilic and nucleophilic character, respectively, during the carbon–nitrogen bond-forming step. In their work, the researchers showed the activation of amines and nitroalkanes with an electrophilic iodine source to directly make amide products. The suggested mechanism showed that the polarities of the two reactants were reversed during carbon–nitrogen bond formation relative to traditional approaches. Looking forward, the researchers noted that using nitroalkanes as acyl anion equivalents provides a conceptually innovative approach to amide and peptide synthesis, and one that may further engender more efficient peptide synthesis that relies on enantioselective methods (7).

"Scientists from many disciplines have sought improved methods to streamline the synthesis of peptides through purely chemical means in order to increase the diversity of the chemical tools available for the design of improved therapeutics," said Jeff Johnston, professor of chemistry at Vanderbilt University, in a June 23, 2010, Vanderbilt University press release. "Our discovery of a conceptually new approach to peptide synthesis brings this capability much closer to reality."

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


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