MEET US AT CEDARLANE EXPO
CedarLane Expo is Toronto’s premier life science industry show. This year’s event will be held on May 23, at the MaRS Centre, South Tower Auditorium, 101 College Street, Toronto.
Come to this free event to see what’s new in the fields of stem cells, immunology, molecular biology, virology, diabetes, obesity, cancer research, and more. Talk directly with suppliers and discuss your projects with the product experts of Bachem.
Bachem supports its customers in the pursuit of groundbreaking discoveries that further scientific advances, particularly in the field of medicine. A comprehensive catalog of biochemicals deliverable ex-stock, an exclusive custom syntheses service for research labs and a full range of services to the pharma and biotech industries complete our service portfolio. We invite you to visit us at our Booth: we are excited to meet with you and learn your needs for peptides. Let us discuss how Bachem can assist to advance your research!
We look forward to meeting you at CedarLane Expo 2019!
CYCLIC PEPTIDES
Cyclic peptides play key roles in various processes and have excellent potential as therapeutics. Examples of cyclic peptide drugs are the hormone oxytocin and the immunosuppressant cyclosporine A, which are both depicted in Figure 1.
Figure 1 Structures of Oxytocin (left) and Cyclosporine A (right).
Cyclic peptides possess several favorable properties as therapeutics. They display a large surface area, which leads to high affinity and selectivity for protein targets and their limited conformational flexibility reduces the entropic penalty upon binding, which in turn improves their binding properties.
The structures shown in Figure 1 each represent an example of the two most common methods of peptide cyclization: amide bond formation, with cyclosporine A being an example, and disulfide bond formation, with oxytocin being an example.
Cyclization via amide bond formation is possible between a free amine group and a free acid functionality in the peptide chain. There are three possible ways for this to proceed:
- The cyclization proceeds between the N- and the C-terminus of the peptide chain (“head-to-tail cyclization”)
- The N-terminus reacts with a free acid functionality of a side chain (e.g. Glu, Asp), or the C-terminus reacts with a side chain bearing a free amine group (e.g. Lys)
- The cyclization takes place between two side chains (e.g. between Lys and Glu)
These three cases are represented schematically in Figure 2:
Figure 2: Possible Cyclization Scenarios through Amide Bond Formation
Orthogonality of Protecting Groups
In order to form one of the cases depicted in Figure 2 selectively, the peptide needs to be in a very specific state of functional group protection. All potentially reactive functional groups need to be protected, except the two, which are intended to form the cyclizing linkage. In the case of amide bond formation, this requires all undesired acid functionalities (e.g. side chains of Glu and Asp) as well as amine functionalities (e.g. side chain of Lys) to be protected. Cyclization is then performed by activating the free acid functionality, which reacts with the free amine moiety.
The situation for disulfide bond formation is similar. If more than two free thiol groups are present upon oxidation, there is a risk, that an undesired disulfide bond connectivity is formed.
In order to arrive at the selectively deprotected peptide sequence, special protecting groups are necessary, which behave orthogonally to the protecting groups already employed during SPPS (e.g. Fmoc/tBu). Introduction of such protecting groups is considered to add a “dimension” to the orthogonality of the protecting group scheme. An overview on possible levels of orthogonality for amide bond formation is depicted in Table 1.
| Orthogonality “Dimension” | Examples for feasible combinations of protecting groups | ||
|---|---|---|---|
| 2nd | Fmoc/tBu | ||
| 3rd | Fmoc/tBu/allyl Fmoc/tBu/Mmt(1) Fmoc/tBu/Dde(1) | ||
| 4th | Fmoc/tBu/allyl/Dde Fmoc/tBu/Mmt/allyl | ||
| 5th | Fmoc/tBu/allyl/Dde/Mmt Fmoc/tBu/allyl/Dde/Bzl(1,2) Fmoc/tBu/Mmt/allyl/Dde | ||
| 6th | Fmoc/tBu/Mmt/allyl/Dde/Bzl |
For more information on the orthogonality of protecting groups, please consult our monograph Orthogonality.
The selective deprotection of cysteine residues is possible as well, allowing for the same type of orthogonality as depicted in Table 1.
In order to enable our partners to perform such selective deprotections, Bachem offers a wide array of Fmoc-Cysteines and Boc-Cysteines bearing different protecting groups. For the optimal deprotection and oxidation conditions, please consult our monograph Cysteine Derivatives.
Cyclization Strategies
The cyclization step is perhaps the most important step in the synthesis of cyclic peptides. There are several different strategies for performing it, but most of them can be grouped into one of two categories – cyclization in solution or cyclization on a solid support (so called “on resin cyclization”). Solution phase cyclizations are well-established reactions and work especially well if the conformation of the peptide in solution can be shifted close to the final conformation prior to cyclization (i.e. by the incorporation of a pseudoproline moiety – see Figure 3).
Figure 3 Synthesis of a Head-to-Tail Cyclized Pentapeptide is aided by Conformational Changes Caused by the incorporation of a Pseudoproline.
As depicted in Figure 3, one of the main side reactions of solution phase cyclization is the oligomerization of the peptide chains. To avoid this reactivity, the reaction has to be performed in very dilute conditions, which favor the intramolecular reactivity over the intermolecular one. In larger scale reactions, such high dilutions are generally unfavored due to the large amount of solvent needed and the slow reaction rates.
On-resin cyclizations can generally be performed at higher concentrations, due to the pseudo-dilution effect. This effect occurs, since most of the immobilized peptide chains are not in close proximity and this prevents them from reacting with each other. This allows for cyclization reactions, which can be performed with lower amounts of solvent and in a shorter time. Additionaly, on-resin reactions can be performed in synthesizers, which allows for high-throughput screening of sequences, e.g. in the search for new drug candidates.
Other Types of Peptide Cycles
Apart from the previously mentioned amide and disulfide bond formation, there are numerous other methods of peptide cyclization, which are increasingly in the focus of drug development activities.
Substitution of sulfur in disulfide bonds with other heteroatoms (e.g. selenium) or carbon has been reported, leading to linkages such as Se-Se, CH2-S or CH2-CH2. Different carbon based linkers are also being employed. The so-called stapled peptides can be prepared via transition metal catalyzed ring-closing metathesis from peptides containing unnatural amino acids, such as O-allyl serine or other amino acids bearing terminally unsaturated hydrocarbon chains. Another cyclization method involving unsaturated functionalities are 1,3-dipolar cycloadditions, often referred to as click chemistry.
Peptides containing an aliphatic azide and an alkyne functionality can be cyclized in a Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) under benign reaction conditions. Our monograph Click Chemistry contains more information on CuAAC as well as metal-free alternatives. Linkage over ester functionalities is also possible, leading to depsipeptides.
Cyclic peptides hold a plethora of possibilities for drug discovery. We offer numerous cyclic peptides as part of our catalog, including
- Antimicrobial peptides, e.g. Defensin Peptides and Baceridin
- Hormones and their agonists, e.g. Oxytocin and Carbetocin
- Toxins, e.g. ω-Conotoxin GVIA and ShK
- Bicyclic peptides, e.g. Phalloidin
- RGD Peptides, e.g. Cilengitide and Galacto-RGD
We also offer cyclization of peptides as part of our custom synthesis service and in the development of new chemical entities (NCEs).
References
M.A.Abdalla, L.J.McGaw, Natural cyclic peptides as an attractive modality for therapeutics: A mini review. Molecules 2018, 23
M.Eder, S.Pavan, U.Bauder-Wüst, K.van Rietschoten, A.-C.Baranski, H.Harrison et al, Bicyclic peptides as a new modality for imaging and targeting of proteins overexpressed by tumors. Cancer Research 2019, 29, 841-852
N.Jain, S.H.Friedman, A tetra-orthogonal strategy for the efficient synthesis of scaffolds based on cyclic peptides. International Journal of Peptide Research and Therapeutics 2018, 24, 535-542
M.Muttenthaler, Å.Andersson, I.Vetter, R.Menon, M.Busnelli, L.Ragnarsson et al, Subtle modifications to oxytocin produce ligands that retain potency and improved selectivity across species. Science Signalling 2017, 10, eaan3398
L.D.Walensky, G.H.Bird, Hydrocarbon-stapled peptides: principles, practice, and progress. Journal of Medicinal Chemistry 2014, 57, 6275-6288
A.Zorzi, K.Deyle, C.Heinis, Cyclic peptide therapeutics: past, present and future. Current Opinion in Chemical Biology 2017, 38, 24-29
CYCLIC PEPTIDES IN CLINICAL DEVELOPMENT
Naturally occurring cyclic peptides continue to be an inspiration for peptide drug design due to their resistance to enzyme degradation and enhanced protein binding affinity. Cyclic peptides often have better biological activity compared to linear peptides due to conformational rigidity (1). In recent years, this class of molecules has been propelled forward by the development of new methods to improve the drug-like properties of peptides by constraining their structure and the creation of new platforms to screen large libraries of cyclic molecules (2). In 2018, Aplidin® (plitidepsin), a synthetic cyclic depsipeptide originally of marine origin, was approved by the Australian Regulatory Agency for the treatment of multiple myeloma (3). There are several cyclic peptides currently in clinical development for a variety of indications as highlighted in Table 1 below.
| Product Name | Generic Name | Indication | Highest Phase | Company |
|---|---|---|---|---|
| Vyleesi | bremelanotide | Erectile Dysfunction, Female Sexual Dysfunction, Female Hypoactive Sexual Desire Disorder | Pre-registration | Palatin Technologies Inc |
| APL-2 | pegcetacoplan | Paroxysmal Nocturnal Hemoglobinuria, Geographic Atrophy, Autoimmune Hemolytic Anemia, Complement-dependent nephropathies | III | Apellis Pharmaceuticals Inc |
| AZP-531 | livoletide | Obesity, Type 2 Diabetes, Ischemia, Prader-Willi Syndrome (PWS) | III | Millendo Therapeutics Inc |
| POL7080 | murepavadin | Cystic Fibrosis, Bronchiectasis, Ventilator Associated Pneumonia (VAP), Hospital-Acquired Pneumonia (HAP), Pseudomonas Infections | III | Polyphor AG |
| BMN 111 | vosoritide | Achondroplasia | III | BioMarin Pharmaceutical Inc |
| ALRN-6924 | -- | Retinoblastoma, Lymphoma, Acute Lymphocytic Leukemia (ALL, Acute Lymphoblastic Leukemia) | II | Aileron Therapeutics Inc |
| BT-1718 | -- | Breast Cancer, Sarcomas, Solid Tumor, Squamous Non-Small Cell Lung Cancer | II | Bicycle Therapeutics Ltd |
| RA-101495 | zilucoplan | Lupus Nephritis, Myasthenia Gravis, Paroxysmal Nocturnal Hemoglobinuria, Atypical Hemolytic Uremic Syndrome | II | Ra Pharmaceuticals Inc |
| CEND-1 | -- | Breast Cancer, Ovarian Cancer, Prostate Cancer, Glioblastoma Multiforme (GBM), Non-Small Cell Lung Cancer | II | DrugCendR Inc |
| NP-213 | novexatin | Onychomycosis (Tinea Unguium) | II | Novabiotics Ltd |
| POL6014 | -- | Chronic Obstructive Pulmonary Disease (COPD), Cystic Fibrosis, Bronchiectasis, Pulmonary Arterial Hypertension, Acute Lung Injury | II | Santhera Pharmaceuticals Holding AG |
| POL6326 | balixafortide | Myocardial Infarction, Asthma, Hematopoietic Stem Cell Transplantation, Inflammation, Metastatic Breast Cancer | I | Polyphor AG |
| ShK-186 | dalazatide | Multiple Sclerosis, Inflammatory Bowel Disease, Lupus Nephritis, Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis (ANCA Vasculitis), Psoriatic Arthritis | I | Kv1.3 Therapeutics |
| THR-149 | -- | Diabetic Macular Edema | I | Oxurion NV |
Pre-registration
VyleesiTM (bremelanotide) is a cyclic peptide derivative being developed by Palatin Technologies for female sexual arousal disorder (FSAD). Vyleesi is a synthetic analog of the naturally occurring hormone α-Melanocyte-Stimulating Hormone (α-MSH) which targets the melanocortin 4 receptor. In 2018, Palatin’s North American licensee for bremelanotide, AMAG Pharmaceuticals, submitted a New Drug Application (NDA) to the U.S. Food and Drug Administration (FDA) for Vyleesi for the treatment of hypoactive sexual desire disorder in premenopausal women (3).
Phase III Clinical Candidates
Apellis Pharmaceuticals is developing APL-2 for the treatment of paroxysmal nocturnal hemoglobinuria, geographic atrophy, autoimmune hemolytic anemia and other indications. APL-2 is a cyclic peptide conjugated to a polyethylene glycol (PEG) polymer and targets complement factor C3, a protein of the immune system. In 2019, the FDA granted Fast Track designation to APL-2 for the treatment of patients with paroxysmal nocturnal hemoglobinuria (3).
AZP-531 (livoletide), a cyclic peptide analog of unacylated ghrelin, is under development by Millendo Therapeutics for the treatment of Prader Willi syndrome and ischemia-related diseases. AZP-531 acts as a growth hormone secretagogue receptor type 1 agonist. In 2019, Millendo Therapeutics initiated a pivotal Phase IIb/III clinical study to investigate livoletide in patients with Prader Willi syndrome (3).
Polyphor is developing POL7080 (murepavadin), a member of a new class of antibiotics, for the treatment of ventilator-associated pneumonia and hospital-acquired pneumonia caused by Pseudomonas aeruginosa (3). Murepavadin is a 14-mer cyclic peptide which targets the outer membrane protein of Pseudomonas aeruginosa (4). In 2019, Polyphor announced the enrollment of the first patient in a Phase III clinical trial for murepavadin for the treatment of ventilator-associated bacterial pneumonia due to Pseudomonas aeruginosa (3).
BioMarin Pharmaceutical is developing BMN 111 (vosoritide) for the treatment of achondroplasia, the most common form of dwarfism. Vosoritide is an analog of a small cyclic peptide known as C-type natriuretic peptide (CNP), a positive regulator of bone growth. The analog peptide is an atrial natriuretic peptide receptor 2 agonist. By the end of 2019, BioMarin expects that top line results will be available from their current Phase III study of vosoritide in children (3).
Conclusion
Several cyclic peptides are in advanced phases of clinical development and many others are in earlier phases. Cyclic peptides are an enticing class of molecules as they often exhibit improved bioactivity and in many cases overcome challenges associated with linear peptides such as poor oral availability, membrane permeability and metabolic stability. For researchers and companies engaged in the research and development of cyclic peptides, Bachem offers a comprehensive Custom Synthesis Service and production of New Chemical Entities.
References
(1) J.Sang Hoon, Cyclic Peptides as Therapeutics Agents and Biochemical Tools. Biomol. Ther. (Seoul). 20, 19-26 (2012)
(2) C.Cain, Excited About Cycling. BioCentury. (2012)
(3) GlobalData (2019)
(4) Murepavadin (POL7080). Polyphor (2019)
MEET BACHEM: MARIA RODRIGUEZ-WELSH
What is your official job title at Bachem?
Assistant Project Management
How long have you been with Bachem? Where did you work before Bachem?
I have been with Bachem for 6 months. Before, I worked for the North Carolina Biotechnology Center and the North Carolina State University in the USA.
Briefly, what do you do at Bachem?
I support the Project Management team mainly by entering data and generating reports in salesforce, attending and writing meeting minutes, and assisting with various project tasks.
What is your academic background?
I have a bachelor in Microbiology and a master in Plant Biology. I also have a Project Management Professional (PMP) certification.
What do you like to do outside of work?
I enjoy spending time with my family, traveling, cooking, and reading.
What do you like most about your job?
I like learning about the differences in complexity of the projects and how each project team functions. I enjoy working with people who are not only technically knowledgeable but are also kind and have a good sense of humor.
What do you do for fun?
Besides the activities I mentioned before, I am learning German!
Thank you very much Maria.
Peptide highlights
Interesting news about peptides in basic research and pharmaceutical development:
Peptides with brominated tryptophan analogs could protect marine animals-EurekAlert!
Peptide shows promise for protecting kidneys from nephritis-Augusta University
New approach to treat eye cancer-European Biotechnology
Experimental drug delivers one-two punch to vision loss-Science Daily
LITERATURE CITATIONS
Bachem peptides and biochemicals are widely cited in research publications. Congratulations to all our customers with recent publications!
R.Jia et al.
A sea anemone-inspired small synthetic peptide at sub-ppm concentrations enhanced biofilm mitigation.
International Biodeterioration & Biodegradation 139, 78-85 (2019)
P.Salgado-Mejias et al.
Structure of Rhoptry Neck Protein 2 is essential for the interaction in vitro with Apical Membrane Antigen 1 in Plasmodium vivax.
Ö.Tezgel et al.
An alternative approach to create N-substituted cyclic dipeptides.
Polymer Chemistry 10, 776-785 (2019)
G.Weltrowska et al.
Equipotent enantiomers of cyclic opioid peptides at μ opioid receptor.