Laboratory of Biochemistry, College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, Republic of Korea
There are many cyclic peptides with diverse biological activities, such as antibacterial activity, immunosuppressive activity, and anti-tumor activity, and so on. Encouraged by natural cyclic peptides with biological activity, efforts have been made to develop cyclic peptides with both genetic and synthetic methods. The genetic methods include phage display, intein-based cyclic peptides, and mRNA display. The synthetic methods involve individual synthesis, parallel synthesis, as well as split-and-pool synthesis. Recent development of cyclic peptide library based on split-and-pool synthesis allows on-bead screening, in-solution screening, and microarray screening of cyclic peptides for biological activity. Cyclic peptides will be useful as receptor agonist/antagonist, RNA binding molecule, enzyme inhibitor and so on, and more cyclic peptides will emerge as therapeutic agents and biochemical tools.
Cyclic peptides are polypeptide chains taking cyclic ring structure. The ring structure can be formed by linking one end of the peptide and the other with an amide bond, or other chemically stable bonds such as lactone, ether, thioether, disulfide, and so on. N-to-C (or head-to-tail) cyclization is amide bond formation between amino and carboxyl termini, and many biologically active cyclic peptides are formed this way. Several cyclic peptides found in nature are used in clinic. The examples are gramicidin and tyrocidine with bactericidal activity, cyclosporin A with immunosuppressive activity, and vancomycin with antibacterial activity, and so on. While peptides have been generally considered to be poor drug molecules, there are some advantages of peptide drugs too. Followings are the weakness of peptides and the strength will be discussed afterward. First, per oral absorption is poor for peptide drugs. In most cases, the route of administration is injection as peptides are not well absorbed in the gastrointestinal tract. Second, peptides are rapidly metabolized, even after successful absorption, by proteolytic enzymes. Third, peptides usually do not cross cell membrane as some small molecules do. If the target of a peptide drug is in the cytoplasm, the peptide may not even reach the target. In spite of these limitations, peptides can be good alternatives to small synthetic molecules because of following advantages. Compared to small synthetic molecules, peptides possess less toxicity and they would not accumulate in organs. Even the fact that peptides get degraded rapidly can be a good thing. Peptide drugs can be less harmful, after acting on target molecules, as they will disappear rapidly by proteolytic degradation. The degradation products are simply amino acids and would not have toxicity (Loffet, 2002). Peptides can work on their targets very selectively, as the interaction with the targets is very specific compared to small molecules (Hummel
Usually, cyclic peptides show better biological activity compared to their linear counterparts due to the conformational rigidity (Edman, 1959; Horton
To test this, a group of peptides has been synthesized, and their cell permeability was compared between cyclic and linear peptides. The results indicated that a peptide does not cross the membrane better simply because it is cyclized (Kwon and Kodadek, 2007). If a certain cyclic peptide is membrane permeable, it is because there are structural features allowing the molecule to cross the cell membrane.For example, cyclosporin A has several intra-molecular hydrogen bonds keeping hydrophilic groups from the surface of the molecule. Overall, structural rigidity, receptor selectivity, biochemical stability are general features of cyclic peptides and some cyclic peptides can be membrane permeable. These features allow cyclic peptides to be good therapeutic agents or biochemical tools, and efforts have been made to develop synthetic cyclic peptide with biological activity. In this review, the role of cyclic peptides in therapeutics and biochemistry will be described, as well as the approaches to develop cyclic peptide compounds for such purposes.
Tyrocidine is a cyclodecapeptide with antibacterial activity. It was found from a culture extract of a soil bacillus,
Cyclosporin A ([R-[R*,R*- (E)]]-cyclic (L-alanyl-D-alanyl-N-methyl-L-leucyl-N-methyl-L-leucyl-N-methyl-L-valyl-3-hy-droxy-N,4-dimethyl-L-2-amino-6-octenoyl-L-α-aminobutyryl-N-methylglycyl-N-methyl-L-leucyl-L-valyl-N-methyl-L-leucyl) is a cyclic peptide isolated from a fungus
For cells to interact with extracellular matrix, integrins play
an important role mediating signals from both in and out of cells. A functional integrin unit comprises of two subunits, α and β subunits in various combinations of isotypes. Many integrins recognize the tripeptide sequence -Arg-Gly-Asp- (RGD) for the interaction with the extracellular ligands. Interestingly, it was found that peptides containing RGD could inhibit tumor cell growth (Humphries
We can find several cyclic peptides from natural peptide hormones such as calcitonin, oxytocin, somatostatin, vasopressin, and so on. These peptides form rigid structure by forming disulfide bond connecting two Cys residues in the peptide.
As described above, there are many cyclic peptides used in clinic, and most of them originate from the natural cyclic peptides. As several features make cyclic peptides attractive lead compounds for drug development as well as nice tools for biochemical research, scientists made diverse efforts to develop biologically active cyclic peptide compounds. Peptides can be prepared by either genetic or synthetic method. The genetic method, as described below, is usually limited to ribosomal 20 amino acids, whereas the sequence determination of hit compounds is straightforward. The synthetic method can provide more versatile cyclic peptide compounds as the repertoire of amino acids and the way of forming cyclic peptides is diverse. Solid-phase peptide synthesis combined with split-and-pool synthesis (Furka
In the following paragraphs, both genetic and synthetic approaches to develop cyclic peptide compounds will be discussed.
Phage display technology was not initially designed to develop cyclic peptide compounds when it was introduced first (Smith, 1985). In this technology, each phage particle displays unique peptide on its surface and the hit can be selected for binding toward a target molecule. Usually, peptides are displayed on the N-terminus, middle, or C-terminus of coat proteins, and the peptide sequence from each phage particle is directed by the DNA sequence of the same phage particle, allowing easy sequence determination. The screening can be repeated as long as encoding DNA molecules are preserved, and this repeated procedure, called bio-panning, is used to enrich the best binders. The diversity of phage display method can be typically up to billions (-109) of phages. As mentioned above, the peptide displayed on the surface of phage particle was not meant to be cyclic peptide. However, cyclic peptides formed from disulfide bond formation were sometimes obtained from phage display as exampled by RGD peptide (Koivunen
peptides are nonribosomal, and they are not accessible with phage display.
mRNA display is an
As mentioned already, cyclic peptides can be synthesized by solid-phase synthesis in addition to typical organic synthesis. Until recently, the modification or improvement of the cyclic peptides involved individual chemical synthesis. This process is very time consuming to extract the structure and activity relationship. Compared to the theoretical diversity for a cyclodecapeptide (over 1013 from 2010, assuming 20 ribosomal amino acids are used), the practical diversity from individual synthesis is very little. Typically, scientists have tried to modify a couple of positions in a cyclic peptide to obtain better, improved compounds with the diversity not exceeding hundreds. When individual compounds are synthesized separately from the beginning to the end, the process is called sequential synthesis. When the synthetic intermediates are split during the synthesis and separate vessels are used for later steps, the process is called the parallel synthesis. In either case, it is difficult to prepare a large size cyclic peptide library. One nice example of synthetic approach can be seen in the development of cyclic RGD peptide. As described above, RGD peptide has affinity toward many integrins which play an important role in angiogenesis. Kessler (Mas-Moruno
While Kessler and coworkers relied on individual synthesis, there are other efforts with higher throughput. Walsh and co-workers employed an enzyme domain from non-ribosomal peptide synthetase for cyclization of precursor synthesized on solid phase (Kohli
Another remarkable approach was the one taken by Guo and co-workers (Qin
As described above, synthetic approach allows the incorporation of non-ribosomal amino acids in the cyclic peptide. However, the diversity that could be obtainable from the ge-netic approach cannot be reached with the sequential parallel synthesis. Split-and-pool synthesis allows the preparation of peptide libraries in large scales, and many linear peptide libraries have been prepared and screened for biological applications. Preparation of cyclic peptide libraries by split-and-pool synthesis is not difficult in terms of synthesis. The bottle-neck is the sequence identification after the hit is selected. Recent advancement in high-throughput sequence determination allows the peptide sequence determination for hundreds micro-beads in a short time, compared to automated Edman analysis which analyzes each bead individually. During partial Edman degradation process, the portion of peptides is kept intact with the help of capping reagents while the peptides are degraded by PITC. At the end of the degradation, each bead yields a ladder of peptides for mass analysis (Sweeney and Pei, 2003; Thakkar
In the previous paragraphs, cyclic peptides with clinical applications, the genetic and synthetic approaches to develop cyclic peptide compounds were discussed. Now, potential applications of cyclic peptides as biochemical tools will be discussed.
Structural rigidity combined with diverse peptide sequence can provide a binding motif toward target molecules. Compared to small molecules, they can be more selective while the size of molecule can be smaller than protein molecules such as antibodies and growth factors. The RGD peptide shown above can be a good example of cyclic peptide as receptor binding molecule. We can find more examples in which cyclic peptides work on receptors. One nice approach from Park and coworkers was to synthesize a mimetic of a monoclonal antibody specific for the p185HER2/neu growth factor receptor (Park
Gene expression can be regulated in multiple levels, and RNA can be a target of regulation as seen in RNA interference (RNAi). The use of cyclic peptides can be one way to modulate RNA activity or stability as shown by Varani and co-workers (Athanassiou
While the above example was limited to certain type of RNA, it would be nice to have cyclic peptide molecules binding to the RNA of interest. This would allow us temporal regulation of gene expression with stable peptide compounds.
As explained above, cyclosporin A inhibits phosphatase activity of calcineurin to exert its immunosuppressive action. Among naturally occurring cyclic peptides, there are protease inhibitors such as sunflower trypsin inhibitor-1 (sfti-1) comprising in 14 amino acids (Colgrave
Cyclic peptides have several structural features making them good drug leads, and there are several naturally occurring cyclic peptides in clinical use. In addition, biologically active cyclic peptides have been developed with genetic and synthetic approaches and they are useful as therapeutics and biochemical tools. With the introduction of new high throughput screening methods, there will be more cyclic peptides working as receptor agonists/antagonists, RNA binding molecules, and enzyme inhibitors.