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European Biopharmaceutical Review

Best in Class

Drugs approved by the Food and Drug Administration and other regulatory agencies worldwide fall under two main categories: small molecules and biologics, or large proteins. Both of these classes experience limitations that restrict their functional range. Small molecules target human proteins bearing lipophilic pockets on their surface, while large proteins and antibodies target through the exterior of the cell. Thus, both of these drug classes do not interact with the targets inside the cells.

Both small molecules and larger proteins together can access only 25 per cent of cell targets, while the other 75 per cent of human proteins inside the cell are not targeted by these two classes of drugs. As a result, there is a great deal of interest among researchers in targeting these sites through the development of a novel therapeutic class, such as bicyclic peptides, that lies between the small molecule and monoclonal antibody (mAb) drug classes.

Bicyclic Advantages

Cyclic peptides are polypeptide chains, and their amino and carboxyl termini are linked together with a peptide bond that forms a circular chain. Bicyclic peptides are polypeptides forming two circular units that have a higher binding affinity and selectivity to protein targets. The introduction of disulfide bridges provides rigidity to the molecule and increases its drug-like properties. These peptides also have a number of advantages over larger protein-based ligands in terms of chemical synthesis, better tissue penetration and a wider choice of application routes (1).

Bicyclic peptides can potentially combine the favourable properties of both major classes of molecules, and may bind as tightly and specifically as antibodies, while being small enough to diffuse into tissues. Due to their relatively rigid conformation, they can bind with high affinity and specificity to protein targets. Unlike linear peptides, they are significantly more stable and resistant to proteases in the blood circulation.

Based on the type of bonds in the cyclic and bicyclic peptides, they are classified into two groups:

  • Homodetic (cyclic and bicyclic) peptides contain bonds between the alpha carboxyl of one residue and the alpha amine of another, such as in cyclosporine A and malformin A
  • Heterodetic peptides contain disulfide bonds, as well as covalent linkages with non-peptide molecules, such as acyl groups in oxytocin and vancomycin

Depending on their cyclisation topology, homodetic peptides can be further classified as:

  • Head-to-tail, where the formation of the ring takes place by cyclisation between functional terminals of the amino and carboxyl group (malformin A)
  • Side chain to side chain: the cyclisation connects the side chain functional groups (α-amanitin)
  • Side chain to end: amino or carboxyl group from the side chain links with the ‘N’ or ‘C’ terminal of the amino acid – such as in cyclic isopeptides – containing at least one nonalpha amide linkage, for example in microcystin
  • Backbone to backbone: amide nitrogens of the peptide backbone are connected through a bridge with alkyl groups and an amide bond
  • Branched: peptide chains are connected through lactams

Depsipeptides are peptides in which one of the amide linkages is replaced by a lactone linkage. An example of depsipeptide is aureobasidin A. Some cyclic and bicyclic depsipeptides are cyclised between the C-terminal carboxyl and the side chain of a threonine or serine residue. Bicyclic peptides such as the amatoxins (amanitin) and phalloidin contain a bridging group, generally between two of the side chains. There are a number of cyclic peptide hormones that are cyclised through a disulfide bond between two cysteines – for example, somatostatin and oxytocin.

Phage Methods

There are several ways to generate potent bicyclic peptides, the most common being phage-encoded libraries. Phage strategies have been developed for disease targets and the generation of potent phage followed by cyclisation of the phage-encoded linear peptides containing three cysteine residues (2). The strategy was successfully applied on human plasma kallikrein inhibitors generated with nanomolar affinities (3,4).

Synthesis of cyclic and bicyclic peptides is complex. Bicyclic peptides have been prepared by solution phase and solid phase synthesis. Higher levels of orthogonality and regioselectivity of amides are crucial while designing the synthetic schemes for bicyclic peptides (5).

The solid phase synthetic approach towards bicyclic peptides has two protection schemes that are widely used: Boc/Bzl strategy (adapted with the presence of sensitive groups) and the Fmoc/tert-Butyl chemistry, which uses piperidine and trifluoroacetic acid respectively to remove these protecting groups. The first approach has been successfully applied on thousands of peptides; the second has been useful in introducing the orthogonality required in these complex bicyclic peptides.

This is followed by a slow cyclisation step which requires excess proportionality of reagents and optimisation of conditions (6). Phosphonium salts, which can be used in excess without side reactions, are preferred over aminium reagent, as the excess use of aminium can lead to the formation of guanidinio derivatives. Solid phase cyclisations are carried out with PyBOP/HOBt/DIEA for 60 minutes.This progress can be monitored by ninhydrin, matrix-assisted laser desorption/ionisation and high-performance liquid chromatography spectra.

Penetration and Diffusion

mAbs have shown high affinity and specificity toward biological structures with minimal damage to normal healthy cells. They have proved efficient in the treatment of severe and chronic diseases such as cancer and autoimmune conditions. The fact that half of the top-selling drugs in 2011 and 2012 fall in this category underlines the importance of this class of drugs. The biggest limitation for these mAbs is their large size, which makes them complicated to administer to patients, thus restricting their efficient diffusion into tissues. Bicyclic or polycyclic peptides can mimic large mAbs, but they are much smaller in size.

These bicyclic and polycyclic peptides may have better penetration and diffusion properties with wider choices of administration than mAbs. They offer better target specificity with increased ability to disrupt protein-protein interactions. The only caveat here is the difficulty in generating polycyclic peptides with suitable binding properties.

Some of the bicyclic peptides in clinical trials are with three to six amino acids in each loop and are 40-100Å in size – ideal for intercepting protein-protein interactions, probably with high affinity. Since they are smaller than the antibodies, these peptides would easily penetrate into target tissues. Matrix metalloproteinase 14 (MMP14), involved in tumour metastasis is a good example. It has been demonstrated that bicyclic peptides screened against MMP14 takes 40-60 minutes for tissue penetration, whereas mAbs take 12-20 hours to penetrate the tumour tissue (7).

Industry Use

Radiolabeled somatostatin analogues have been used to target somatostatin receptor (sst) tumours. Recently developed bicyclic somatostatin-based radiopeptides are a new type of radiotherapy for neuroendocrine tumours and can be used as diagnostic imaging agents. These new analogues showed high affinity for sst2 and sst3 and moderate affinity towards sst1, sst4 and sst5 (8). The analogue AM3, comprising an octreotide ring and a head-to-tail coupled arg-diaminobutyric acid ring, showed the highest receptor affinity and agonist potency. The pharmacokinetic data of 177Lu/68Ga-AM3 make this peptide an excellent candidate for imaging and as a positron emission tomography radiotracer.

Another class of bicyclic peptides in the early stages of development are compounds targeting arginine-glycineaspartic acid vectors that bind to the receptors associated with angiogenesis-like integrin receptors. These bicyclic compounds may be used for the diagnosis or treatment of malignancy, heart disease, endometriosis, inflammation-related diseases, rheumatoid arthritis and Kaposi’s sarcoma. The patent WO 03/006491 describes peptide-based compounds targeting integrin receptors associated with angiogenesis. The European EP1648925 patent describes a new class of the bicyclic peptide and the analogues used as imaging agents (9).

Other classes of bicyclic peptides include:

Histone Acetyl Transferase and Histone Deacetyalse (HDAC)

These enzymes take care of balancing the dynamic process of acetylation and deacetylation of the amino groups of lysine at the N-terminal tails of the core histones. Imbalance in this process can bring about changes in the transcriptional regulation of genes involved in important functions like the differentiation and apoptosis processes. This may disrupt the acetylation and deacetylation process, which in turn can lead to carcinogenesis. Inhibition of HDAC enzymatic activity is expected to induce re-expression of differentiation-inducing genes (10).

Plasma Kallikrein

Bicyclic peptide antagonists of the serine protease plasma kallikrein inhibitors have potential application in hereditary angioedema – a life-threatening disease characterised by local swelling in subcutaneous tissue and organs (3).


Cyclic peptides can bind and transport metal cations across membranes in biological systems. Monocyclic peptides are too flexible and non-specifically bind to metal ions, whereas bicyclic peptides are more rigid and specifically bind cations. A series of bicyclic peptides have been designed and synthesised by researchers to provide ammonium ion complexation sites via hydrogen bonding in a tetrahedral geometry. This helps to determine the accuracy levels of ammonium ions in the presence of potassium ions for the clinical evaluation of urea and creatinine levels used in blood analysis applications (11).

Bioactive Bicyclic Peptides

Bicyclic peptides are forming an important class of therapeutic agents with marketed products, such as actinomycin,endothelin, vancomycin and deoxybouvardin. A number of bicyclic compounds are in early developmental stages that therapeutically target HIV (cycloviolin) and tumours.

The bicyclic octapeptide moroidin is a natural product found in stinging nettles (Urtica dioica) and is responsible for pain and rashes on the skin (12). Moroidin has a strong anti-mitotic activity, as it inhibits tubulin polymerisation, thus interfering with cell division (see Figure 2, page 36).

Peptide Toxins

Another category of bicyclic peptides are peptide toxins. These are capable of causing injury and disease when in contact with body tissues, as they interact with biomolecules such as enzymes, hormones and cellular receptors. Toxins vary greatly in their severity, ranging from usually minor or acute reactions to fatality.

Some examples of bicyclic peptide toxins include:

Theonellamide F

A novel antifungal bicyclic peptide from the marine sponge Theonella induces formation of vacuoles and overproduction of 1, 3-beta-D-glucan. Theonellamide F inhibits growth of various pathogenic fungi and has cytotoxic properties against L1210 and P388 leukaemia cells (13). It can be used as important biomarker for the measurement of antigens and as a non-invasive method for detecting invasive fungal infections.


A bicyclic heptapeptide that binds tightly to actin filaments and prevents their depolarisation. Actin polymerisation and depolymerisation are necessary for chemotaxis and cytokinesis in cells. Fluorescent phalloidin derivatives are useful in identifying the distribution of F-actin in cells.


This bicyclic peptide of eight amino acids is the most deadly of all the toxins found in several species of the Amanita genus of mushrooms. It is a useful tool in determining types of ribonucleic acid polymerase present in a tissue sample.

VEGF Peptide Inhibitors

Researchers at the Ludwick Institute of Cancer Research in Germany have patented small, conformationally-constrained peptide inhibitors of the vascular endothelial growth factor (VEGF) family. Their possible clinical applications include inhibition of the angiogenesis, lymphangiogenesis and neovascularisation to inhibit cancer growth, diabetic retinopathy and other chronic conditions. Dimeric bicyclic peptide inhibitors based on the sequences of exposed loops of growth factor proteins act as protein growth inhibitors and are useful anti-tumour agents (14). These bicyclic peptides interfere with the action of VEGF-D and VEGF-C, or any one protein, and prevent activation to help stop the growth of cancer.

Excellent Candidates

Bicyclic peptides are an emerging class of drug molecules with some of the favourable properties of both small molecules and mAbs. The size (macrocyclic loops) and functionality of bicyclic peptides provide better specifi city, binding properties, rigidity, stability and good tissue penetration. Bicyclic peptides are excellent potential therapeutic drug candidates that have low toxic profiles.


1. Baeriswyl V and Heinis C, Polycyclic peptide therapeutics, Chem Med Chem 8(3): pp377-384, 2013

2. Rentero Rebollo I and Heinis C, Phage selection of bicyclic peptides, Methods 15, 60(1): pp46-54, 2013

3. Baeriswyl V et al, Bicyclic peptides with optimized ring size inhibit human plasma kallikrein and its orthologues while sparing paralogous proteases, Chem Med Chem 7(7): pp1,173-1,176, 2012

4. Heinis C et al, Phage-encoded combinatorial chemical libraries based on bicyclic peptides, Nat Chem Biol 5(7): pp502-507, 2009

5. Teixidó M et al, Bicyclic homodetic peptide libraries: comparison of synthetic strategies for their solid-phase synthesis, J Comb Chem 5: pp760-768, 2003

6. Davies JS, The cyclisation of peptides and depsipeptides, J Peptide Sci 9: pp471-501, 2003

7. Nandra K, OBR Review, Thursday 16th May 2013

8. Fani M et al, Radiolabeled bicyclic somatostatin-based analogs: a novel class of potential radiotracers for SPECT/PET of neuroendocrine tumors, J Nucl Med 51: pp1,771-1,779

9. Cuthbertson A and Solbakken M, Imaging agents: World Patent WO 03/006491, European Patent EP1648925

10. Islam NM, Kato T, Nishino N, Kim HJ, Ito A and Yoshida M, Bioorganic & Medicinal Chemistry Letters 20: pp997-999, 2010

11. AI N, Keller A, Kolker E and Aebersold R, Anal Chem 75(17): pp4,646-4,658, 2003

12. Morita H, Shimbo K, Shigemori H and Kobayashi J, Bioorganic & Medicinal Chemistry Letters 10(5): pp469-471, 2000

13. Matsunaga S et al, J Am Chem Soc 11(1): pp2,582-2,588, 1989

14. Achen M, Cendron A, Hughes R and Stacker S, US patent: US 20020065218 A1, European Patent: EP 1248642 A1

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Archana Gangakhedkar is a Technical Marketing Specialist at American Peptide Company, Inc. She has an MS in Organic Medicinal Chemistry from Osmania University and is a certifi cate holder in Regulatory Affairs from UCSC Extension. Prior to joining the company, Archana was a Research Scientist at Xenoport and is the inventor of several new chemical entities.
Archana Gangakhedkar
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