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Discovery and applications of naturally occurring cyclic peptides
Drug Discovery Today: Technologies
Vol. 9, No. 1 2012
Editors-in-Chief Kelvin Lam – Harvard University, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Peptides or modified peptides as drug molecules
Discovery and applications of naturally occurring cyclic peptides L. Thorstholm, D.J. Craik* Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia
Over the last decade several families of naturally occurring cyclic peptides have been discovered that are extremely stable and have important roles as defense molecules for their host organisms. Because of their
Section Editors: Greg Verdine – Harvard University, Cambridge, MA, USA. Sihyun Ham – Sookmyung Women’s University, Seoul, Republic of Korea.
exceptional stability and potent bioactivities they can be adapted for use as scaffolds in drug development. Here we describe technologies for the application of cyclic peptides in drug design. Introduction Our focus here is on naturally occurring cyclic peptides that are ribosomally synthesized and thus composed of the 20 proteinogenic amino acids. They occur in all kingdoms of life, as highlighted in Fig. 1, and range in size from approximately 12 to 80 amino acids. In addition to having a head-to-tail cyclic backbone, many of these peptides contain one or more disulfide bonds. Features common to these peptides include that they are ultra-stable and typically have a role in host defense of their producing organisms, for example via the expression of antimicrobial or pesticidal activities. Because of their exceptional stability they are able to act in harsh biological milieu and this advantage, over conventional (acyclic) peptides, provides opportunities for potential applications in drug design. In this article we give an overview of the origins and structures of the currently known naturally occurring cyclic peptides and describe technologies for adapting them for drug development purposes.
Discovery of naturally occurring cyclic peptide The naturally occurring head-to-tail cyclic peptides discussed here have been discovered over the past 15 years in bacteria, *Corresponding author.: D.J. Craik ([email protected]) 1740-6749/$ ß 2011 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2011.07.005
fungi, plants and animals [1,2], as summarized in Fig. 1. These ribosomally synthesized cyclic peptides contrast with earlier known cyclic peptides such as cyclosporin, which typically contain modified amino acids and are synthesized in microorganisms by non-ribosomal peptide synthetases (NRPS) [3]. Table 1 gives an overview of examples of ribosomally synthesized cyclic peptides recorded in CyBase (http://www.cybase. org.au/), a database dedicated to cyclic peptides and circular proteins. Initial reports on the sequences and structures of naturally occurring cyclic peptides occurred in the 1990s [4–7]. Some foreshadowing of their potential therapeutic applications had earlier occurred with reports that one of the peptides, kalata B1, isolated from an African plant called kalata kalata (Oldenlandia affinis), had effects on uterine contractions when used as a medicinal tea by Congolese women during labor [8]. The active ingredient was characterized as a very stable polypeptide, but the structure of the backbone cyclized peptide was not determined until 1995 [9]. Other cyclic peptides have now been found in a wide range of organisms.
Cyclic peptides in bacteria Ribosomally synthesized cyclic peptides produced in bacteria contain 35–78 amino acids and are often hydrophobic in character. They occur in Gram-positive bacteria as a subclass of bacteriocins, and in Gram-negative bacteria as pilins (Table 1). The unifying feature of the bacteriocins is their e13
Drug Discovery Today: Technologies | Peptides or modified peptides as drug molecules
(a)
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Amanita phalloide
Helianthus annus
Macaca mulatta
Oldenlandia affinis
Momordica cochinchinensis Enteroccoccus faecalis
7
14
18
29
34
Alpha-amanitin
SFTI-I
RTD-1
Kalata B1
MCoTI-II
(b)
(c)
70
(d)
AS-48 Drug Discovery Today: Technologies
Figure 1. Naturally occurring cyclic peptides. (a) Names and images of organisms in which cyclic peptides are expressed. (b) Schematic representations of the cyclic backbone and intramolecular connections involving disulfide bonds are shown in orange. The cysteine arrangement of the three disulfide bonds in kalata B1 and MCoTI-II are in a cystine knot and those of RTD-1 are in a cystine ladder. Note that alpha-amanitin does not have a disulfide bond, but rather a covalent crosslink between cysteine and tryptophan. (c) Shows the number of residues in the cyclic backbone. (d) Three dimensional ribbon representation of selected cyclic peptides.
antimicrobial activity. The host organism is immune to the peptides it expresses, which typically act in defense against other microorganisms. This antimicrobial activity has led to applications in the food industry, and one cyclic peptide in particular, AS-48, is intensively used for food preservation because of its activity against salmonella and listeria, and its thermal stability [10]. Among the most recent bacterial cyclic peptides to be discovered are lactocyclicin Q and garvicin ML isolated from cheese and Mallard ducks, respectively. Both peptides are extremely stable and have antimicrobial activity [11,12].
Cyclic peptides in fungi Ribosomally expressed cyclic peptides from fungi have been reported only in the past few years. Typically they are small, containing only seven or eight amino acids, and differ from other cyclic peptides by having an internal cross-link which is not a disulfide bond. Two classes of cyclic peptides discovered recently in fungi, exemplified by alpha-amanitin and phallocidin are highly toxic and target RNA polymerase II and F-actin, respectively [13].
Cyclic peptides in plants Cyclic peptides from plants can be divided into two classes: Sunflower trypsin inhibitors and cyclotides [14]. Sunflower e14
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trypsin inhibitor-1 (SFTI-1) is a 14-residue peptide with one disulfide bond that is extracted from the seeds of the common sunflower, and is a potent trypsin inhibitor. Cyclotides are the largest class of cyclic plant peptides, and have been found in more than 20 species from the Violaceae, Rubiaceae, Cucurbitaceae and Fabaceae plant families. Approximately 200 sequences have been reported so far (http://www.cybase.org.au/), but it has been predicted that the cyclotide class might comprise more than 50,000 members [15]. Cyclotides are 28–37 amino acids in size and have a cystine knotted arrangement of three disulfide bonds. Two of the largest cyclotides, MCoTI-I and MCoTI-II, are also trypsin inhibitors, and are found in the seeds of a bitter melon from Vietnam (Fig. 1).
Cyclic peptides in animals Some primates express cyclic peptides called u-defensins as a part of their immune system. These antimicrobial peptides are expressed in white blood cells and some members of the family have activity against HIV-1 [16]. They comprise 18 amino acids structured in a ring divided by three ‘laddered’ disulfide bonds between six cysteine residues (Fig. 1), and are the only cyclic peptides currently known in mammals. The udefensins were first discovered in 1999 when rhesus theta defensin-1 (RTD-1) was isolated from macaque leukocytes [7].
e15
Peptidec
AA
Sequenced
Activity/commentse
Yearf
Bacteria Agrobacterium tumefaciens
t pilin
74
Expressed by Gram-negative bacteria
1999
Bacillus subtilise Butyrivibrio fibrisolvens
subtilosin A butyrivibriocin AR10
35 58
Anti-bacterial Anti-bacterial
2004 2003
Carnobacterium maltaromaticum
carnocyclin-A
60
Anti-microbial, isolated from pork
2008
Clostridium beijerinckii
circularin A
69
Anti-bacterial, isolated from feces and soil
2003
Enterococcus faecalis
AS-48
70
trbC
78
Anti-bacterial, preservative in the food industry Expressed by Gram-negative bacteria
2000
Escherichia coli Lactobacillus gasseri LA39
gassericin A
58
Lactococcus sp. QU 12
lactocyclicin Q
61
Lactococcus garvieae
garvicin ML
60
Streptococcus uberis
uberolysin
70
QSAGGGTDPATMVNNICTFILGPFGQSLAVLGIVAIGISW MFGRASLGLVAGVVGGIVIMFGASFLGKTLTGGG NKGCATCSIGAACLVDGPIPDFEIAGATGLFGLWG IYFIADKMGIQLAPAWYQDIVNWVSAGGTLTTGFAIIV GVTVPAWIAEAAAAFGIASA LVAYGIAQGTAEKVVSLINAGLTVGSIISILGGVTVGLSG VFTAVKAAIA KQGIKKAIQL VAGALGVQTAAATTIVNVILNAGTLVTVLGIIASIASGGAG TLMTIGWATFKATVQKLAKQSMARAIAY MAKEFGIPAAVAGTVLNVVEAGGWVTTIVSILTAVGSGGL SLLAAAGRESIKAYLKKEIKKKGKRAVIAW SEGTGGSLPYESWLTNLRNSVTGPVAFALSIIGIVVAGGVLI FGGELNAFFRTLIFLVLVMALLVGAQNVMSTFFGRG IYWIADQFGIHLATGTARKLLDAMASGASLGTAFAAILGV TLPAWALAAAGALGATAA LIDHLGAPRW AVDTILGAIA VGNLASWVLA LVPGPGWAVK AGLATAAAIV KHQGKAAAAA W LVATGMAAGVAKTIVNAVSA GMDIATALSL FSGAFTAAGG IMALIKKYAQ KKLWKQLIAA LAGYTGIASGTAKKVVDAIDKGAAAFVIISIISTVISAG ALG AVSASADFIILTVKNYISRNLKAQAVIW
Fungi Amanita phalloides Amanita phalloides
alpha-amanitin phallacidin
Plants Chassalia discolour Chassalia parvifolia Gloeospermum Blakeanum Gloeospermum pauciflorum Helianthus annus Hybanthus floribundus E Hybanthus floribundus W Hybanthus parviflorus Leonia cymosa Melicytus ramiflorus Momordica cochinchinensis Oldenlandia affinis Palicourea condensata Psychotria leptothyrsa Psychotria longipes Psychotria suterella Viola abyssinica
CD-1 circulin A globa A globa A SFTI-1 hyfl A hyfl I hypa A cycloviolin A mram 1 MCoTI-I kalata B1 palicourein psyle A cyclopsychotride A PS-1 vaby A
8 7 34 30 30 32 14 31 30 30 31 31 34 29 37 28 31 31 29
1999
Anti-bacterial, isolated from feces of a human infant Anti-microbial, isolated from cheese
1998
Anti-bacterial, extracted from mallard duck Anti-bacterial, found in bovine
2011
IWGIGCNP AWLVDCP
Toxic Toxic
2007 2007
GADGFCGESCYVIPCISYLVGCSCDTIEKVCKRN GIPCGESCVWIPCISAALGCSCKNKVCYRN GIPCGESCVFIPCITAAIGCSCKTKVCYRN GGSIPCIETCVWTGCFLVPGCSCKSDKKCYLN GRCTKSIPPICFPD SISCGESCVYIPCTVTALVGCTCKDKVCYLN GIPCGESCVFIPCISGVIGCSCKSKVCYRN GIPCAESCVYIPCTITALLGCSCKNKVCYN GVIPCGESCVFIPCISAAIGCSCKNKVCYRN GSIPCGESCVYIPCISSLLGCSCKSKVCYKN GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSD GLPVCGETCVGGTCNTPGCTCSWPVCTRN GDPTFCGETCRVIPVCTYSAALGCTCDDRSDGLCKRN GIACGESCVFLGCFIPGCSCKSKVCYFN SIPCGESCVFIPCTVTALLGCSCKSKVCYKN GFIPCGETCIWDKTCHAAGCSCSVANICVRN GETCAGGTCNTPGCSCSWPICTRNGLPVC
Not reported Hemolytic activity, anti-bacterial, anti-HIV Not reported Not reported Trypsin inhibitor. Anti-cancer activity Not reported Not reported Not reported Anti-HIV Predicted from precursor Trypsin inhibitor Insecticidal, anti-HIV, hemolytic Anti-HIV Not reported Hemolytic, neurotensin inhibitor Not reported Not reported
2008 1994 2010 2010 1999 2005 2005 2001 2000 2009 2000 1995 2001 2010 1994 2008 2011
2009
2007
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Speciesb
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Table 1. Overview of naturally occurring cyclic peptidesa
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Speciesb
Peptidec
AA
Sequenced
Activity/commentse
Yearf
Viola Viola Viola Viola Viola Viola Viola Viola Viola
varv peptide B viba 1 vibi A vico A cycloviolacin H1 cycloviolacin O1 cycloviolacin T1 tricyclon A cycloviolacin Y1
30 30 29 31 30 30 29 33 33
GLPVCGETCFGGTCNTPGCSCDPWPMCSRN GIPCGEGCVYLPCFTAPLGCSCSSKVCYRN GLPVCGETCFGGTCNTPGCSCSYPICTRN GSIPCAESCVYIPCFTGIAGCSCKNKVCYYN GIPCGESCVYIPCLTSAIGCSCKSKVCYRN GIPCAESCVYIPCTVTALLGCSCSNRVCYN GIPVCGETCVGGTCNTPGCSCSWPVCTRN GGTIFDCGESCFLGTCYTKGCSCGEWKLCYGTN GGTIFDCGETCFLGTCYTPGCSCGNYGFCYGTN
Not reported Not reported Not reported Not reported Not reported Molluscicidal, nematocidal Not reported Hemolytic Anti-HIV, hemolytic, nematocidal
1999 2009 2008 2003 1999 2006 2010 2005 2007
RTD-1 BTD-1 PhTD-1 Retrocyclin-1
18 18 18 18
RCICTRGFCRCLCRRGVC RCVCTRGFCRCVCRRGVC RCVCRRGVCRCVCTRGFC RCICGRGICRCICGRGIC
Anti-microbial, anti-bacterial, anti-HIV Anti-microbial Anti-microbial Only expressed on mRNA level
1999 2008 2010 2002
arvensis baoshanensis biflora cotyledon hederaceae odorata tianshanica tricolor yedoensis5
Primates Macaca mulatta Papio anubis Papio hamadryas Homo sapiens a
All data were extracted from the database of cyclic proteins http://www.cybase.org.au/. Species in which ribosomally expressed cyclic peptides are found. Many organisms express multiple cyclic peptides and just one representative example is shown in such cases. Some individual cyclic peptides are expressed in multiple organisms, for example, kalata B1 and gassericin A (also called reutericin 6). d Sequences of cyclic peptides; cysteines involved in disulfide bonding are bold. e Reported biological activity of the peptide. f The year the peptide was classified as a naturally occurring ribosomally expressed cyclic peptide. b c
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Table 1 (Continued )
Table 2. Comparison of technologies for cyclic peptide production Technology 1
Technology 2
Isolation
Chemical synthesis
Technology 3 Biological synthesis
2a
2b
3a
3b
Name of specific type of technology
Isolation of native peptide from organism
Solid phase peptide synthesis (SPPS)
Chemo-enzymatic synthesis
Recombinant expression in plants or plant cells
Recombinant expression in bacteria
Pros
Produces large quantities One organism often contains multiple cyclic peptides
Easy to make sequence modifications Easy to incorporate non native amino acids
Chain can be assembled with Fmoc chemistry Easy to make sequence modifications
High yields possible Using a native expression system to generate genetically modified cyclic peptides
Possible to make libraries of modified cyclic peptides Possible to label with 15N and
Cons
Only native sequences produced Purification of overlapping components can be difficult
So far mainly restricted to Boc chemistry, which require HF cleavage from the resin Can be challenging to obtain native fold
Requires an enzymatic ligation site to be incorporated into the sequence Two step ligation process
In early phase development Limited yields in current systems
Require cysteine residue at the N-terminal and a a-thioester group at the C-terminal
References
[14,24]
[28,29,40]
[20,32]
[27,33,34]
[35–39]
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Drug Discovery Today: Technologies | Peptides or modified peptides as drug molecules
Recently, naturally occurring isoforms of RTD-1 were isolated from baboon leukocytes [17,18]. Humans, lowland gorillas, bonobo and the common chimpanzee possess pseudo-genes encoding u-defensins but do not actively express the cyclic peptides themselves [16].
Structures and activities of naturally occurring cyclic peptides Naturally occurring cyclic peptides vary greatly in structure and size, from the smallest cyclic peptides in fungi containing only 7 residues to the largest in bacteria comprising 78 residues (Fig. 1). However, there are several common properties of these peptides, including well-defined structures, ultrahigh stability and bioactivities that have a pivotal role in defense of the host organism. The host defense properties protect bacteria against competition from other microorganisms, fungi and plants from predators, and animals against diseases. Table 1 lists some of the bioactivities that have been reported in cyclic peptides.
Cyclic peptides and pharmaceutical applications Because of their stability and favorable biopharmaceutical properties, cyclic peptides have a range of potential applications in drug design and development. They are bioactive in their own right and can therefore be used as drug leads but also are excellent scaffolds for the stabilization of foreign bioactive peptide epitopes, and have been used to inspire cyclization of a range of acyclic bioactive peptides.
Direct drug leads The natural functions of cyclic peptides can in some cases lend themselves to applications in drug development. For example, SFTI-1, the natural function of which is thought to inhibit the digestive enzyme trypsin to protect seeds, has
(a)
(b)
high sequence similarity to other serine protease inhibitors, and has also been found to inhibit matriptase, an enzyme implicated as a target in breast cancer [19]. Another example of direct drug leads is the u-defensins, some of which inhibit the entry of HIV-1 into cells. In humans, u-defensins are present only as pseudo genes, which putatively encode cyclic peptides called retrocyclins but do not actually express them. A recent study demonstrated that the retrocyclin gene can be reactivated, via read-through of a premature stop codon, to express retrocyclin peptides [16]. This finding offers potential new approaches to the treatment of HIV and other viral diseases via the re-awakening of defense peptides that have been dormant for around 7 million years.
Grafting of bioactive epitopes onto cyclotide scaffolds Cyclic peptides can be utilized as ultra-stable frameworks, into which linear peptide epitopes can be grafted. In these applications a loop of the native cyclic peptide framework is substituted with a bioactive epitope, as illustrated in Fig. 2. The stability of the linear epitope is enhanced by this incorporation, which therefore enhances the biopharmaceutical properties of the linear peptide. In one example of this approach, an inhibitor of a foot-and-mouth disease virus (FMDV) protease was developed on the basis of the MCoTI-II scaffold [20]. Another study demonstrated that an antagonist of vascular endothelial growth factor-A (VEGF-A) can be stabilized by grafting it into the cyclotide kalata B1 [21].
Re-engineering natural peptides into cyclic peptides Knowledge of naturally occurring cyclic peptides can also be used in to engineer linear peptides into cyclic, more stable,
(c)
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Figure 2. Concept of grafting a peptide epitope into a cyclic peptide scaffold. (a) Peptide epitopes of different conformations are illustrated by different colors. (b) Cyclotide used as grafting scaffold. (c) Modified cyclic peptides, in which the peptide epitopes are grafted into one of the native cyclic peptide loops.
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(a) C CHH2Cl 3O 2 H
(b)
(a)
(b)
(c)
(a)
(b)
Drug Discovery Today: Technologies
Figure 3. Schematic representation of technologies used to obtain cyclic peptides. (a) Technology 1: native peptide extraction, here illustrated by solvent extraction from leaves. After finely grinding the leaves, cyclic peptides are extracted with methanol/dichloromethane. After concentration of the methanol fraction the cyclic peptides are purified by RP-HPLC, and each peak is collected and analyzed by mass spectrometry. (b) Technology 2: chemical synthesis. The C- and N-termini are highlighted with red and blue circles, respectively. Linking the termini can be achieved either chemically (Technology 2a) or enzymatically (Technology 2b). In the final product the peptide backbone is cyclic, and termini no longer exist (purple circle). (c) Technology 3: recombinant expression. A DNA sequence encoding a modified cyclic peptide is incorporated into an external organism (the DNA modification is illustrated by a purple dot), for example a plant (Technology 3a) or bacterium (Technology 3b). The DNA gel illustrates that the host organism is expressing the cyclic peptide (marked with a purple circle), where after the expression is scaled up. The cyclic peptide is obtained after purification with RP-HPLC. The DNA modification results in a change in the amino acid sequence (indicated by a purple dot).
analogs. Specifically, linear peptides that have their C- and N-termini close to each other can be cyclized to increase stability. Two recent examples are the cyclization of a conotoxin, to produce a peptide with potential in the treatment of pain [22], and the cyclization of a chlorotoxin, used as an imaging agent to visualize brain tumors [23]. In both cases the cyclic analogs were more stable than their linear counterparts and had improved biopharmaceutical properties.
Technologies for cyclic peptide production There are three key technologies for the production of cyclic peptides: isolation from host organisms, chemical synthesis, and biological synthesis utilizing recombinant expression systems such as bacteria or plants (Table 2). e18
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Technology 1: Isolation from host organisms The oldest technology for the production of cyclic peptides is direct isolation from a host organism. A recent report described how cyclic peptides from plants are isolated and characterized [24], but a similar approach can be applied to cyclic peptides from other organisms. Figure 3a gives a schematic representation of the isolation technique for cyclotides. First the plant material (e.g. leaves, seeds or roots) is ground and dissolved in a dichloromethane–methanol mixture that is separated on addition of water. The methanol extract is purified through several rounds of C18 column/ HPLC [24], analyzed using mass spectrometry [25] and structurally characterized using NMR spectroscopy [26]. This isolation approach is useful for both the discovery of new sequences and the production of known peptides. Gene
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Drug Discovery Today: Technologies | Peptides or modified peptides as drug molecules
sequencing is increasingly used to discover new cyclic peptide sequences and, conveniently, is an approach that is possible for ribosomally synthesized cyclic peptides but not nonribosomal peptides [17,27].
between cysteine residues has an important role and it can be challenging to obtain the correct connectivity/folding if the peptide contains more than one disulfide bond and thus has the potential to form multiple isomers.
Technology 2: Chemical synthesis
Technology 3: Biological synthesis
The relatively small size of many naturally occurring cyclic peptides makes them suitable for production using solid phase peptide synthesis (SPPS). In brief, the linear peptide precursor is assembled from the C- to N-terminus, with the starting C-terminal residue linked to a solid resin support. Amino acids are assembled in the sequence one by one, with the resin washed with dimethylformamide (DMF) between reaction steps. During assembly of the linear peptide chain, protecting groups are required to prevent unwanted couplings. Two main approaches for amino group protection involve t-butoxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) protecting groups, which are removed by trifluoroacetic acid (TFA) or 20% piperidine in DMF, respectively, in successive cycles of deprotection, wash, coupling and wash during peptide chain assembly. After addition of the final residue, the peptide is cleaved from the resin; if the peptide was assembled with Boc protection chemistry the cleaving agent is hydrofluoric acid (HF), while Fmoc chemistry utilizes TFA cleavage. After purification of the crude peptide, it is folded and cyclized in an appropriate buffer under basic conditions [28–30]. In terms of design of the linear precursor, there are no limitations on the choice of the C-terminal residue, but to achieve cyclization through the preferred ‘native chemical ligation’ (NCL) reaction [31], the final (i.e. N-terminal) residue must be a cysteine and the C-terminus must be joined to the resin with a thioester linker. The susceptibility of the thioester to base (e.g. piperidine) thus generally restricts the synthesis of cyclic peptides via NCL technology to Boc protection chemistry. However, new approaches using Fmoc protection chemistry are emerging [29], which will allow alternatives to the use of HF/Boc chemistry. A recent study used Fmoc chemistry to make a linear peptide sequence that was then cyclized in a chemo-enzymatic approach [20]. This approach (Fig. 3b) is only possible if the peptide can be engineered with an enzymatic ligation site, but should be applicable to a variety of enzymes. So far it has been demonstrated with trypsin, which was used to cyclize synthetic linear forms of the trypsin inhibitors MCoTI-II [20] and SFTI-1 [32]. A significant advantage of chemical (or chemo-enzymatic) approaches to cyclic peptide production, relative to isolation of native peptides, is the opportunity to make modifications or incorporate non-natural amino acids into the sequence. However, one of the challenges with SPPS is to achieve the native fold of the peptide, especially if alterations to the native sequence are introduced. The disulfide connectivity
Recently, plant [33,34] and bacterial [35] expression systems have been developed to produce cyclic peptides (Fig. 3c). Plants and bacteria express large quantities of cyclic peptides in nature, so the idea of using these expression systems to produce modified cyclic peptides on an industrial scale is, in principle, possible using genetic engineering technology. In this approach, a gene encoding the precursor of the desired cyclic peptide is introduced into the organism, which then expresses and processes the precursor to produce the cyclic peptide. One of the main motivations for using recombinant expression systems is to scale up and lower costs of production, but the approach also introduces the potential to generate libraries of cyclic peptides for drug screening [36–38]. Recombinant expression systems are gaining more popularity as production methods for cyclic peptides, and might in the future be the preferred method of production. Plant-based production of engineered cyclic peptides can be achieved either by expressing them in plant cells [33,34] or in whole plants [27], both of which have been applied for disulfide-containing cyclic peptides. Although bacteria naturally produce some cyclic peptides (e.g. bacteriocin AS-48) they do not produce disulfide-rich peptides such as cyclotides. The most successful approach to making such peptides in bacteria is via intein-based methods where the introduced peptide is cyclized intracellularly by a protein splicing domain [35]. One of the advantages of recombinant expression of cyclic peptides is the possibility of isotopically labeling their nitrogen and carbon atoms by growing the bacteria or plants in 15 N/13C enriched media. Isotropic labeling of cyclic peptides has major advantages for monitoring them in solution by NMR spectroscopy, for example to study interactions with target proteins [39], which have major applications in drug discovery.
Conclusions Ribosomally synthesized cyclic peptides are a recently discovered class of molecules, but have already prompted several interesting drug design applications. Understanding more about these very stable bioactive natural products, and improving techniques for identifying and isolating them are important to gain a better understanding of where, how and why they are expressed. From increased knowledge of cyclic peptides in nature, it might be possible to optimize how they can be used in pharmaceutical applications. Chemical synthesis by SPPS chemistry is an efficient way of making cyclic peptides in the laboratory. Its biggest advantage www.drugdiscoverytoday.com
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is that it is easy to make sequence modifications [40] and also to incorporate non-native amino acids. However, SPPS requires use of HF to cleave peptides from the resin, and in some cases it can be difficult to obtain the native fold, resulting in low yields. The currently preferred method of synthesis requires a cysteine residue at the N-terminus and an a-thioester group at the Cterminus of the synthetic precursor peptide, but because most cyclic peptides are cysteine-rich this is not a major drawback. The chemo-enzymatic approach has similar advantages/disadvantages, as the linear peptide is synthesized by SPPS. However, the use of enzymatic cyclization of the peptide backbone allows the alternative chemical route of using Fmoc chemistry rather than Boc chemistry for assembly of the linear peptide precursor, which avoids the use of HF during cleavage from the solid phase resin. Although this gives the chemo-enzymatic approach an advantage over NCL-based Boc chemistry, the need for an enzyme ligation site introduces some limitations. Furthermore, the chemo-enzymatic approach involves an additional reaction step compared to SPPS with chemical cyclization. Expression of non-native cyclic peptides in plant systems is still in the early stages of development. However, the prospects are promising, and plant expression might take the production of cyclic peptides to an industrial scale in due course. However, more plant and plant cell systems need to be tested and there is a need for a greater understanding of biosynthetic cyclization mechanisms to optimize these expression systems. Bacterial expression systems have been shown to be effective in the expression of the cyclotide MCoTI-I, and several mutants have been generated both by changes to the amino acid sequence [37] and by isotopic labeling with 15N nitrogen [39]. Bacterial expression systems need to be further explored on a wider range of cyclic peptides, but appear to be promising systems for the expression of cyclic peptides. In summary, the three major technologies for cyclic peptide production, that is, isolation, chemical synthesis and biological synthesis, are all complementary and purpose dependent but are still rapidly evolving and promise to facilitate a range of exciting applications of cyclic peptides on the near future.
Conflict of interest DJC is an inventor on patents in the field of cyclotides and other cyclic peptides.
Acknowledgements Work in our laboratory on peptide toxins is supported by grants from the National Health & Medical Research Council (NHMRC; Grant ID nos: APP519734, APP631457 and APP1010552) and the Australian Research Council (DP0984390). DJC is an NHMRC Professorial Research Fellow e20
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(ID no. 569603). LT is a recipient of the Cancer Council Queensland PhD Scholarship.
References 1 Cascales, L. and Craik, D.J. (2010) Naturally occurring circular proteins: distribution, biosynthesis and evolution. Org. Biomol. Chem. 8, 5035–5047 2 Craik, D.J. (2006) Chemistry – seamless proteins tie up their loose ends. Science 311, 1563–1564 3 Walsh, C.T. (2004) Polyketide and nonribosomal peptide antibiotics: modularity and versatility. Science 303, 1805–1810 4 Craik, D.J. et al. (2004) Discovery, structure and biological activities of the cyclotides. Curr. Protein Pept. Sci. 5, 297–315 ¨ ransson, U. et al. (2004) Novel strategies for isolation and 5 Go characterization of cyclotides: the discovery of bioactive macrocyclic plant polypeptides in the Violaceae. Curr. Protein Pept. Sci. 5, 317–329 6 Maqueda, M. et al. (2008) Genetic features of circular bacteriocins produced by Gram-positive bacteria. FEMS Microbiol. Rev. 32, 2–22 7 Tang, Y.Q. et al. (1999) A cyclic antimicrobial peptide produced in primate leukocytes by the ligation of two truncated alpha-defensins. Science 286, 498–502 8 Gran, L. (1973) Oxytocic principles of oldenlandia-affinis. Lloydia 36, 174–178 9 Saether, O. et al. (1995) Elucidation of the primary and 3-dimentional structure of the uterotonic polypeptide kalata B1. Biochemistry 34, 4147–4158 10 Viedma, P.M. et al. (2011) Inhibition of spoilage and toxigenic Bacillus species in dough from wheat flour by the cyclic peptide enterocin AS-48. Food Control 22, 756–761 11 Borrero, J. et al. (2011) Characterization of garvicin ML, a novel circular bacteriocin produced by Lactococcus garvieae DCC43, isolated from Mallard ducks (Anas platyrhynchos). Appl. Environ. Microbiol. 77, 369–373 12 Sawa, N. et al. (2009) Identification and characterization of lactocyclicin Q, a novel cyclic bacteriocin produced by Lactococcus sp. strain QU 12. Appl. Environ. Microbiol. 75, 1552–1558 13 Hallen, H.E. et al. (2007) Gene family encoding the major toxins of lethal Amanita mushrooms. Proc. Natl. Acad. Sci. U.S.A. 104, 19097–19101 14 Craik, D.J. et al. (1999) Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol. 294, 1327–1336 15 Gruber, C.W. et al. (2008) Distribution and evolution of circular miniproteins in flowering plants. Plant Cell 20, 2471–2483 16 Venkataraman, N. et al. (2009) Reawakening retrocyclins: ancestral human defensins active against HIV-1. PLoS Biol. 7, 720–729 17 Garcia, A.E. et al. (2008) Isolation, synthesis, and antimicrobial activities of naturally occurring theta-defensin isoforms from baboon leukocytes. Infect. Immun. 76, 5883–5891 18 Stegemann, C. et al. (2010) De novo sequencing of two new cyclic thetadefensins from baboon (Papio hamadryas) leukocytes by matrix-assisted laser desorption/ionization mass spectrometry. Rapid Commun. Mass Spectrom. 24, 599–604 19 Jiang, S. et al. (2007) Design and synthesis of redox stable analogues of sunflower trypsin inhibitors (SFTI-1) on solid support, potent inhibitors of matriptase. Org. Lett. 9, 9–12 20 Thongyoo, P. et al. (2008) Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides. Org. Biomol. Chem. 6, 1462–1470 21 Gunasekera, S. et al. (2008) Engineering stabilized vascular endothelial growth factor-A antagonists: synthesis, structural characterization, and bioactivity of grafted analogues of cyclotides. J. Med. Chem. 51, 7697–7704 22 Clark, R.J. et al. (2005) Engineering stable peptide toxins by means of backbone cyclization: stabilization of the alpha-conotoxin MII. Proc. Natl. Acad. Sci. U.S.A. 102, 13767–13772 23 Akcan, M. et al. (2011) Chemical re-engineering of chlorotoxin improves bioconjugation properties for tumor imaging and targeted therapy. J. Med. Chem. 54, 782–787 24 Ireland, D.C. et al. (2010) Isolation, sequencing, and structure–activity relationships of cyclotides. J. Nat. Prod. 73, 1610–1622
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Poth, A.G. et al. (2011) Discovery of cyclotides in the Fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins. ACS Chem. Biol. 6, 345–355 Daly, N.L. et al. (2011) NMR and protein structure in drug design: application to cyclotides and conotoxins. Eur. Biophys. J. Biophys. Lett. 40, 359–370 Mylne, J.S. et al. (2011) Albumins and their processing machinery are hijacked for cyclic peptides in sunflower. Nat. Chem. Biol. 7, 257–259 Clark, R.J. and Craik, D.J. (2010) Native chemical ligation applied to the synthesis and bioengineering of circular peptides and proteins. Biopolymers 94, 414–422 Park, S. et al. (2010) An efficient approach for the total synthesis of cyclotides by microwave assisted Fmoc-SPPS. Int. J. Pept. Res. Therapeut. 16, 167–176 Tam, J.P. et al. (1999) An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc. Natl. Acad. Sci. U.S.A. 96, 8913–8918 Dawson, P.E. et al. (1994) Synthesis of proteins by native chemical ligation. Science 266, 776–779 Marx, U.C. et al. (2003) Enzymatic cyclization of a potent Bowman-Birk protease inhibitor, sunflower trypsin inhibitor-1, and solution structure of an acyclic precursor peptide. J. Biol. Chem. 278, 21782–21789
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Dornenburg, H. (2010) Cyclotide synthesis and supply: from plant to bioprocess. Biopolymers 94, 602–610 34 Seydel, P. et al. (2009) Scale-up of Oldenlandia affinis suspension cultures in photobioreactors for cyclotide production. Eng. Life Sci. 9, 219–226 35 Camarero, J.A. et al. (2007) Biosynthesis of a fully functional cyclotide inside living bacterial cells. Chembiochem 8, 1363–1366 36 Austin, J. et al. (2010) In vivo biosynthesis of an Ala-scan library based on the cyclic peptide SFTI-1. Amino Acids 38, 1313–1322 37 Austin, J. et al. (2009) Biosynthesis and biological screening of a genetically encoded library based on the cyclotide MCoTI-I. Chembiochem 10, 2663– 2670 38 Sancheti, H. and Camarero, J.A. (2009) ‘Splicing up’ drug discovery. Cellbased expression and screening of genetically-encoded libraries of backbone-cyclized polypeptides. Adv. Drug Deliv. Rev. 61, 908–917 39 Puttamadappa, S.S. et al. (2010) Backbone dynamics of cyclotide MCoTI-I free and complexed with trypsin. Angew. Chem. Int. Ed. Engl. 49, 7030– 7034 40 Huang, Y.H. et al. (2010) Lysine-scanning mutagenesis reveals an amendable face of the cyclotide kalata B1 for the optimization of nematocidal activity. J. Biol. Chem. 285, 10797–10805
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Editors-in-Chief Kelvin Lam – Harvard University, USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Peptides or modified peptides as drug molecules
Discovery and applications of naturally occurring cyclic peptides L. Thorstholm, D.J. Craik* Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia
Over the last decade several families of naturally occurring cyclic peptides have been discovered that are extremely stable and have important roles as defense molecules for their host organisms. Because of their
Section Editors: Greg Verdine – Harvard University, Cambridge, MA, USA. Sihyun Ham – Sookmyung Women’s University, Seoul, Republic of Korea.
exceptional stability and potent bioactivities they can be adapted for use as scaffolds in drug development. Here we describe technologies for the application of cyclic peptides in drug design. Introduction Our focus here is on naturally occurring cyclic peptides that are ribosomally synthesized and thus composed of the 20 proteinogenic amino acids. They occur in all kingdoms of life, as highlighted in Fig. 1, and range in size from approximately 12 to 80 amino acids. In addition to having a head-to-tail cyclic backbone, many of these peptides contain one or more disulfide bonds. Features common to these peptides include that they are ultra-stable and typically have a role in host defense of their producing organisms, for example via the expression of antimicrobial or pesticidal activities. Because of their exceptional stability they are able to act in harsh biological milieu and this advantage, over conventional (acyclic) peptides, provides opportunities for potential applications in drug design. In this article we give an overview of the origins and structures of the currently known naturally occurring cyclic peptides and describe technologies for adapting them for drug development purposes.
Discovery of naturally occurring cyclic peptide The naturally occurring head-to-tail cyclic peptides discussed here have been discovered over the past 15 years in bacteria, *Corresponding author.: D.J. Craik ([email protected]) 1740-6749/$ ß 2011 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2011.07.005
fungi, plants and animals [1,2], as summarized in Fig. 1. These ribosomally synthesized cyclic peptides contrast with earlier known cyclic peptides such as cyclosporin, which typically contain modified amino acids and are synthesized in microorganisms by non-ribosomal peptide synthetases (NRPS) [3]. Table 1 gives an overview of examples of ribosomally synthesized cyclic peptides recorded in CyBase (http://www.cybase. org.au/), a database dedicated to cyclic peptides and circular proteins. Initial reports on the sequences and structures of naturally occurring cyclic peptides occurred in the 1990s [4–7]. Some foreshadowing of their potential therapeutic applications had earlier occurred with reports that one of the peptides, kalata B1, isolated from an African plant called kalata kalata (Oldenlandia affinis), had effects on uterine contractions when used as a medicinal tea by Congolese women during labor [8]. The active ingredient was characterized as a very stable polypeptide, but the structure of the backbone cyclized peptide was not determined until 1995 [9]. Other cyclic peptides have now been found in a wide range of organisms.
Cyclic peptides in bacteria Ribosomally synthesized cyclic peptides produced in bacteria contain 35–78 amino acids and are often hydrophobic in character. They occur in Gram-positive bacteria as a subclass of bacteriocins, and in Gram-negative bacteria as pilins (Table 1). The unifying feature of the bacteriocins is their e13
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(a)
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Amanita phalloide
Helianthus annus
Macaca mulatta
Oldenlandia affinis
Momordica cochinchinensis Enteroccoccus faecalis
7
14
18
29
34
Alpha-amanitin
SFTI-I
RTD-1
Kalata B1
MCoTI-II
(b)
(c)
70
(d)
AS-48 Drug Discovery Today: Technologies
Figure 1. Naturally occurring cyclic peptides. (a) Names and images of organisms in which cyclic peptides are expressed. (b) Schematic representations of the cyclic backbone and intramolecular connections involving disulfide bonds are shown in orange. The cysteine arrangement of the three disulfide bonds in kalata B1 and MCoTI-II are in a cystine knot and those of RTD-1 are in a cystine ladder. Note that alpha-amanitin does not have a disulfide bond, but rather a covalent crosslink between cysteine and tryptophan. (c) Shows the number of residues in the cyclic backbone. (d) Three dimensional ribbon representation of selected cyclic peptides.
antimicrobial activity. The host organism is immune to the peptides it expresses, which typically act in defense against other microorganisms. This antimicrobial activity has led to applications in the food industry, and one cyclic peptide in particular, AS-48, is intensively used for food preservation because of its activity against salmonella and listeria, and its thermal stability [10]. Among the most recent bacterial cyclic peptides to be discovered are lactocyclicin Q and garvicin ML isolated from cheese and Mallard ducks, respectively. Both peptides are extremely stable and have antimicrobial activity [11,12].
Cyclic peptides in fungi Ribosomally expressed cyclic peptides from fungi have been reported only in the past few years. Typically they are small, containing only seven or eight amino acids, and differ from other cyclic peptides by having an internal cross-link which is not a disulfide bond. Two classes of cyclic peptides discovered recently in fungi, exemplified by alpha-amanitin and phallocidin are highly toxic and target RNA polymerase II and F-actin, respectively [13].
Cyclic peptides in plants Cyclic peptides from plants can be divided into two classes: Sunflower trypsin inhibitors and cyclotides [14]. Sunflower e14
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trypsin inhibitor-1 (SFTI-1) is a 14-residue peptide with one disulfide bond that is extracted from the seeds of the common sunflower, and is a potent trypsin inhibitor. Cyclotides are the largest class of cyclic plant peptides, and have been found in more than 20 species from the Violaceae, Rubiaceae, Cucurbitaceae and Fabaceae plant families. Approximately 200 sequences have been reported so far (http://www.cybase.org.au/), but it has been predicted that the cyclotide class might comprise more than 50,000 members [15]. Cyclotides are 28–37 amino acids in size and have a cystine knotted arrangement of three disulfide bonds. Two of the largest cyclotides, MCoTI-I and MCoTI-II, are also trypsin inhibitors, and are found in the seeds of a bitter melon from Vietnam (Fig. 1).
Cyclic peptides in animals Some primates express cyclic peptides called u-defensins as a part of their immune system. These antimicrobial peptides are expressed in white blood cells and some members of the family have activity against HIV-1 [16]. They comprise 18 amino acids structured in a ring divided by three ‘laddered’ disulfide bonds between six cysteine residues (Fig. 1), and are the only cyclic peptides currently known in mammals. The udefensins were first discovered in 1999 when rhesus theta defensin-1 (RTD-1) was isolated from macaque leukocytes [7].
e15
Peptidec
AA
Sequenced
Activity/commentse
Yearf
Bacteria Agrobacterium tumefaciens
t pilin
74
Expressed by Gram-negative bacteria
1999
Bacillus subtilise Butyrivibrio fibrisolvens
subtilosin A butyrivibriocin AR10
35 58
Anti-bacterial Anti-bacterial
2004 2003
Carnobacterium maltaromaticum
carnocyclin-A
60
Anti-microbial, isolated from pork
2008
Clostridium beijerinckii
circularin A
69
Anti-bacterial, isolated from feces and soil
2003
Enterococcus faecalis
AS-48
70
trbC
78
Anti-bacterial, preservative in the food industry Expressed by Gram-negative bacteria
2000
Escherichia coli Lactobacillus gasseri LA39
gassericin A
58
Lactococcus sp. QU 12
lactocyclicin Q
61
Lactococcus garvieae
garvicin ML
60
Streptococcus uberis
uberolysin
70
QSAGGGTDPATMVNNICTFILGPFGQSLAVLGIVAIGISW MFGRASLGLVAGVVGGIVIMFGASFLGKTLTGGG NKGCATCSIGAACLVDGPIPDFEIAGATGLFGLWG IYFIADKMGIQLAPAWYQDIVNWVSAGGTLTTGFAIIV GVTVPAWIAEAAAAFGIASA LVAYGIAQGTAEKVVSLINAGLTVGSIISILGGVTVGLSG VFTAVKAAIA KQGIKKAIQL VAGALGVQTAAATTIVNVILNAGTLVTVLGIIASIASGGAG TLMTIGWATFKATVQKLAKQSMARAIAY MAKEFGIPAAVAGTVLNVVEAGGWVTTIVSILTAVGSGGL SLLAAAGRESIKAYLKKEIKKKGKRAVIAW SEGTGGSLPYESWLTNLRNSVTGPVAFALSIIGIVVAGGVLI FGGELNAFFRTLIFLVLVMALLVGAQNVMSTFFGRG IYWIADQFGIHLATGTARKLLDAMASGASLGTAFAAILGV TLPAWALAAAGALGATAA LIDHLGAPRW AVDTILGAIA VGNLASWVLA LVPGPGWAVK AGLATAAAIV KHQGKAAAAA W LVATGMAAGVAKTIVNAVSA GMDIATALSL FSGAFTAAGG IMALIKKYAQ KKLWKQLIAA LAGYTGIASGTAKKVVDAIDKGAAAFVIISIISTVISAG ALG AVSASADFIILTVKNYISRNLKAQAVIW
Fungi Amanita phalloides Amanita phalloides
alpha-amanitin phallacidin
Plants Chassalia discolour Chassalia parvifolia Gloeospermum Blakeanum Gloeospermum pauciflorum Helianthus annus Hybanthus floribundus E Hybanthus floribundus W Hybanthus parviflorus Leonia cymosa Melicytus ramiflorus Momordica cochinchinensis Oldenlandia affinis Palicourea condensata Psychotria leptothyrsa Psychotria longipes Psychotria suterella Viola abyssinica
CD-1 circulin A globa A globa A SFTI-1 hyfl A hyfl I hypa A cycloviolin A mram 1 MCoTI-I kalata B1 palicourein psyle A cyclopsychotride A PS-1 vaby A
8 7 34 30 30 32 14 31 30 30 31 31 34 29 37 28 31 31 29
1999
Anti-bacterial, isolated from feces of a human infant Anti-microbial, isolated from cheese
1998
Anti-bacterial, extracted from mallard duck Anti-bacterial, found in bovine
2011
IWGIGCNP AWLVDCP
Toxic Toxic
2007 2007
GADGFCGESCYVIPCISYLVGCSCDTIEKVCKRN GIPCGESCVWIPCISAALGCSCKNKVCYRN GIPCGESCVFIPCITAAIGCSCKTKVCYRN GGSIPCIETCVWTGCFLVPGCSCKSDKKCYLN GRCTKSIPPICFPD SISCGESCVYIPCTVTALVGCTCKDKVCYLN GIPCGESCVFIPCISGVIGCSCKSKVCYRN GIPCAESCVYIPCTITALLGCSCKNKVCYN GVIPCGESCVFIPCISAAIGCSCKNKVCYRN GSIPCGESCVYIPCISSLLGCSCKSKVCYKN GGVCPKILKKCRRDSDCPGACICRGNGYCGSGSD GLPVCGETCVGGTCNTPGCTCSWPVCTRN GDPTFCGETCRVIPVCTYSAALGCTCDDRSDGLCKRN GIACGESCVFLGCFIPGCSCKSKVCYFN SIPCGESCVFIPCTVTALLGCSCKSKVCYKN GFIPCGETCIWDKTCHAAGCSCSVANICVRN GETCAGGTCNTPGCSCSWPICTRNGLPVC
Not reported Hemolytic activity, anti-bacterial, anti-HIV Not reported Not reported Trypsin inhibitor. Anti-cancer activity Not reported Not reported Not reported Anti-HIV Predicted from precursor Trypsin inhibitor Insecticidal, anti-HIV, hemolytic Anti-HIV Not reported Hemolytic, neurotensin inhibitor Not reported Not reported
2008 1994 2010 2010 1999 2005 2005 2001 2000 2009 2000 1995 2001 2010 1994 2008 2011
2009
2007
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Speciesb
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Table 1. Overview of naturally occurring cyclic peptidesa
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Speciesb
Peptidec
AA
Sequenced
Activity/commentse
Yearf
Viola Viola Viola Viola Viola Viola Viola Viola Viola
varv peptide B viba 1 vibi A vico A cycloviolacin H1 cycloviolacin O1 cycloviolacin T1 tricyclon A cycloviolacin Y1
30 30 29 31 30 30 29 33 33
GLPVCGETCFGGTCNTPGCSCDPWPMCSRN GIPCGEGCVYLPCFTAPLGCSCSSKVCYRN GLPVCGETCFGGTCNTPGCSCSYPICTRN GSIPCAESCVYIPCFTGIAGCSCKNKVCYYN GIPCGESCVYIPCLTSAIGCSCKSKVCYRN GIPCAESCVYIPCTVTALLGCSCSNRVCYN GIPVCGETCVGGTCNTPGCSCSWPVCTRN GGTIFDCGESCFLGTCYTKGCSCGEWKLCYGTN GGTIFDCGETCFLGTCYTPGCSCGNYGFCYGTN
Not reported Not reported Not reported Not reported Not reported Molluscicidal, nematocidal Not reported Hemolytic Anti-HIV, hemolytic, nematocidal
1999 2009 2008 2003 1999 2006 2010 2005 2007
RTD-1 BTD-1 PhTD-1 Retrocyclin-1
18 18 18 18
RCICTRGFCRCLCRRGVC RCVCTRGFCRCVCRRGVC RCVCRRGVCRCVCTRGFC RCICGRGICRCICGRGIC
Anti-microbial, anti-bacterial, anti-HIV Anti-microbial Anti-microbial Only expressed on mRNA level
1999 2008 2010 2002
arvensis baoshanensis biflora cotyledon hederaceae odorata tianshanica tricolor yedoensis5
Primates Macaca mulatta Papio anubis Papio hamadryas Homo sapiens a
All data were extracted from the database of cyclic proteins http://www.cybase.org.au/. Species in which ribosomally expressed cyclic peptides are found. Many organisms express multiple cyclic peptides and just one representative example is shown in such cases. Some individual cyclic peptides are expressed in multiple organisms, for example, kalata B1 and gassericin A (also called reutericin 6). d Sequences of cyclic peptides; cysteines involved in disulfide bonding are bold. e Reported biological activity of the peptide. f The year the peptide was classified as a naturally occurring ribosomally expressed cyclic peptide. b c
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Table 1 (Continued )
Table 2. Comparison of technologies for cyclic peptide production Technology 1
Technology 2
Isolation
Chemical synthesis
Technology 3 Biological synthesis
2a
2b
3a
3b
Name of specific type of technology
Isolation of native peptide from organism
Solid phase peptide synthesis (SPPS)
Chemo-enzymatic synthesis
Recombinant expression in plants or plant cells
Recombinant expression in bacteria
Pros
Produces large quantities One organism often contains multiple cyclic peptides
Easy to make sequence modifications Easy to incorporate non native amino acids
Chain can be assembled with Fmoc chemistry Easy to make sequence modifications
High yields possible Using a native expression system to generate genetically modified cyclic peptides
Possible to make libraries of modified cyclic peptides Possible to label with 15N and
Cons
Only native sequences produced Purification of overlapping components can be difficult
So far mainly restricted to Boc chemistry, which require HF cleavage from the resin Can be challenging to obtain native fold
Requires an enzymatic ligation site to be incorporated into the sequence Two step ligation process
In early phase development Limited yields in current systems
Require cysteine residue at the N-terminal and a a-thioester group at the C-terminal
References
[14,24]
[28,29,40]
[20,32]
[27,33,34]
[35–39]
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Recently, naturally occurring isoforms of RTD-1 were isolated from baboon leukocytes [17,18]. Humans, lowland gorillas, bonobo and the common chimpanzee possess pseudo-genes encoding u-defensins but do not actively express the cyclic peptides themselves [16].
Structures and activities of naturally occurring cyclic peptides Naturally occurring cyclic peptides vary greatly in structure and size, from the smallest cyclic peptides in fungi containing only 7 residues to the largest in bacteria comprising 78 residues (Fig. 1). However, there are several common properties of these peptides, including well-defined structures, ultrahigh stability and bioactivities that have a pivotal role in defense of the host organism. The host defense properties protect bacteria against competition from other microorganisms, fungi and plants from predators, and animals against diseases. Table 1 lists some of the bioactivities that have been reported in cyclic peptides.
Cyclic peptides and pharmaceutical applications Because of their stability and favorable biopharmaceutical properties, cyclic peptides have a range of potential applications in drug design and development. They are bioactive in their own right and can therefore be used as drug leads but also are excellent scaffolds for the stabilization of foreign bioactive peptide epitopes, and have been used to inspire cyclization of a range of acyclic bioactive peptides.
Direct drug leads The natural functions of cyclic peptides can in some cases lend themselves to applications in drug development. For example, SFTI-1, the natural function of which is thought to inhibit the digestive enzyme trypsin to protect seeds, has
(a)
(b)
high sequence similarity to other serine protease inhibitors, and has also been found to inhibit matriptase, an enzyme implicated as a target in breast cancer [19]. Another example of direct drug leads is the u-defensins, some of which inhibit the entry of HIV-1 into cells. In humans, u-defensins are present only as pseudo genes, which putatively encode cyclic peptides called retrocyclins but do not actually express them. A recent study demonstrated that the retrocyclin gene can be reactivated, via read-through of a premature stop codon, to express retrocyclin peptides [16]. This finding offers potential new approaches to the treatment of HIV and other viral diseases via the re-awakening of defense peptides that have been dormant for around 7 million years.
Grafting of bioactive epitopes onto cyclotide scaffolds Cyclic peptides can be utilized as ultra-stable frameworks, into which linear peptide epitopes can be grafted. In these applications a loop of the native cyclic peptide framework is substituted with a bioactive epitope, as illustrated in Fig. 2. The stability of the linear epitope is enhanced by this incorporation, which therefore enhances the biopharmaceutical properties of the linear peptide. In one example of this approach, an inhibitor of a foot-and-mouth disease virus (FMDV) protease was developed on the basis of the MCoTI-II scaffold [20]. Another study demonstrated that an antagonist of vascular endothelial growth factor-A (VEGF-A) can be stabilized by grafting it into the cyclotide kalata B1 [21].
Re-engineering natural peptides into cyclic peptides Knowledge of naturally occurring cyclic peptides can also be used in to engineer linear peptides into cyclic, more stable,
(c)
Drug Discovery Today: Technologies
Figure 2. Concept of grafting a peptide epitope into a cyclic peptide scaffold. (a) Peptide epitopes of different conformations are illustrated by different colors. (b) Cyclotide used as grafting scaffold. (c) Modified cyclic peptides, in which the peptide epitopes are grafted into one of the native cyclic peptide loops.
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1:1
(a) C CHH2Cl 3O 2 H
(b)
(a)
(b)
(c)
(a)
(b)
Drug Discovery Today: Technologies
Figure 3. Schematic representation of technologies used to obtain cyclic peptides. (a) Technology 1: native peptide extraction, here illustrated by solvent extraction from leaves. After finely grinding the leaves, cyclic peptides are extracted with methanol/dichloromethane. After concentration of the methanol fraction the cyclic peptides are purified by RP-HPLC, and each peak is collected and analyzed by mass spectrometry. (b) Technology 2: chemical synthesis. The C- and N-termini are highlighted with red and blue circles, respectively. Linking the termini can be achieved either chemically (Technology 2a) or enzymatically (Technology 2b). In the final product the peptide backbone is cyclic, and termini no longer exist (purple circle). (c) Technology 3: recombinant expression. A DNA sequence encoding a modified cyclic peptide is incorporated into an external organism (the DNA modification is illustrated by a purple dot), for example a plant (Technology 3a) or bacterium (Technology 3b). The DNA gel illustrates that the host organism is expressing the cyclic peptide (marked with a purple circle), where after the expression is scaled up. The cyclic peptide is obtained after purification with RP-HPLC. The DNA modification results in a change in the amino acid sequence (indicated by a purple dot).
analogs. Specifically, linear peptides that have their C- and N-termini close to each other can be cyclized to increase stability. Two recent examples are the cyclization of a conotoxin, to produce a peptide with potential in the treatment of pain [22], and the cyclization of a chlorotoxin, used as an imaging agent to visualize brain tumors [23]. In both cases the cyclic analogs were more stable than their linear counterparts and had improved biopharmaceutical properties.
Technologies for cyclic peptide production There are three key technologies for the production of cyclic peptides: isolation from host organisms, chemical synthesis, and biological synthesis utilizing recombinant expression systems such as bacteria or plants (Table 2). e18
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Technology 1: Isolation from host organisms The oldest technology for the production of cyclic peptides is direct isolation from a host organism. A recent report described how cyclic peptides from plants are isolated and characterized [24], but a similar approach can be applied to cyclic peptides from other organisms. Figure 3a gives a schematic representation of the isolation technique for cyclotides. First the plant material (e.g. leaves, seeds or roots) is ground and dissolved in a dichloromethane–methanol mixture that is separated on addition of water. The methanol extract is purified through several rounds of C18 column/ HPLC [24], analyzed using mass spectrometry [25] and structurally characterized using NMR spectroscopy [26]. This isolation approach is useful for both the discovery of new sequences and the production of known peptides. Gene
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Drug Discovery Today: Technologies | Peptides or modified peptides as drug molecules
sequencing is increasingly used to discover new cyclic peptide sequences and, conveniently, is an approach that is possible for ribosomally synthesized cyclic peptides but not nonribosomal peptides [17,27].
between cysteine residues has an important role and it can be challenging to obtain the correct connectivity/folding if the peptide contains more than one disulfide bond and thus has the potential to form multiple isomers.
Technology 2: Chemical synthesis
Technology 3: Biological synthesis
The relatively small size of many naturally occurring cyclic peptides makes them suitable for production using solid phase peptide synthesis (SPPS). In brief, the linear peptide precursor is assembled from the C- to N-terminus, with the starting C-terminal residue linked to a solid resin support. Amino acids are assembled in the sequence one by one, with the resin washed with dimethylformamide (DMF) between reaction steps. During assembly of the linear peptide chain, protecting groups are required to prevent unwanted couplings. Two main approaches for amino group protection involve t-butoxycarbonyl (Boc) or 9-fluorenylmethyloxycarbonyl (Fmoc) protecting groups, which are removed by trifluoroacetic acid (TFA) or 20% piperidine in DMF, respectively, in successive cycles of deprotection, wash, coupling and wash during peptide chain assembly. After addition of the final residue, the peptide is cleaved from the resin; if the peptide was assembled with Boc protection chemistry the cleaving agent is hydrofluoric acid (HF), while Fmoc chemistry utilizes TFA cleavage. After purification of the crude peptide, it is folded and cyclized in an appropriate buffer under basic conditions [28–30]. In terms of design of the linear precursor, there are no limitations on the choice of the C-terminal residue, but to achieve cyclization through the preferred ‘native chemical ligation’ (NCL) reaction [31], the final (i.e. N-terminal) residue must be a cysteine and the C-terminus must be joined to the resin with a thioester linker. The susceptibility of the thioester to base (e.g. piperidine) thus generally restricts the synthesis of cyclic peptides via NCL technology to Boc protection chemistry. However, new approaches using Fmoc protection chemistry are emerging [29], which will allow alternatives to the use of HF/Boc chemistry. A recent study used Fmoc chemistry to make a linear peptide sequence that was then cyclized in a chemo-enzymatic approach [20]. This approach (Fig. 3b) is only possible if the peptide can be engineered with an enzymatic ligation site, but should be applicable to a variety of enzymes. So far it has been demonstrated with trypsin, which was used to cyclize synthetic linear forms of the trypsin inhibitors MCoTI-II [20] and SFTI-1 [32]. A significant advantage of chemical (or chemo-enzymatic) approaches to cyclic peptide production, relative to isolation of native peptides, is the opportunity to make modifications or incorporate non-natural amino acids into the sequence. However, one of the challenges with SPPS is to achieve the native fold of the peptide, especially if alterations to the native sequence are introduced. The disulfide connectivity
Recently, plant [33,34] and bacterial [35] expression systems have been developed to produce cyclic peptides (Fig. 3c). Plants and bacteria express large quantities of cyclic peptides in nature, so the idea of using these expression systems to produce modified cyclic peptides on an industrial scale is, in principle, possible using genetic engineering technology. In this approach, a gene encoding the precursor of the desired cyclic peptide is introduced into the organism, which then expresses and processes the precursor to produce the cyclic peptide. One of the main motivations for using recombinant expression systems is to scale up and lower costs of production, but the approach also introduces the potential to generate libraries of cyclic peptides for drug screening [36–38]. Recombinant expression systems are gaining more popularity as production methods for cyclic peptides, and might in the future be the preferred method of production. Plant-based production of engineered cyclic peptides can be achieved either by expressing them in plant cells [33,34] or in whole plants [27], both of which have been applied for disulfide-containing cyclic peptides. Although bacteria naturally produce some cyclic peptides (e.g. bacteriocin AS-48) they do not produce disulfide-rich peptides such as cyclotides. The most successful approach to making such peptides in bacteria is via intein-based methods where the introduced peptide is cyclized intracellularly by a protein splicing domain [35]. One of the advantages of recombinant expression of cyclic peptides is the possibility of isotopically labeling their nitrogen and carbon atoms by growing the bacteria or plants in 15 N/13C enriched media. Isotropic labeling of cyclic peptides has major advantages for monitoring them in solution by NMR spectroscopy, for example to study interactions with target proteins [39], which have major applications in drug discovery.
Conclusions Ribosomally synthesized cyclic peptides are a recently discovered class of molecules, but have already prompted several interesting drug design applications. Understanding more about these very stable bioactive natural products, and improving techniques for identifying and isolating them are important to gain a better understanding of where, how and why they are expressed. From increased knowledge of cyclic peptides in nature, it might be possible to optimize how they can be used in pharmaceutical applications. Chemical synthesis by SPPS chemistry is an efficient way of making cyclic peptides in the laboratory. Its biggest advantage www.drugdiscoverytoday.com
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is that it is easy to make sequence modifications [40] and also to incorporate non-native amino acids. However, SPPS requires use of HF to cleave peptides from the resin, and in some cases it can be difficult to obtain the native fold, resulting in low yields. The currently preferred method of synthesis requires a cysteine residue at the N-terminus and an a-thioester group at the Cterminus of the synthetic precursor peptide, but because most cyclic peptides are cysteine-rich this is not a major drawback. The chemo-enzymatic approach has similar advantages/disadvantages, as the linear peptide is synthesized by SPPS. However, the use of enzymatic cyclization of the peptide backbone allows the alternative chemical route of using Fmoc chemistry rather than Boc chemistry for assembly of the linear peptide precursor, which avoids the use of HF during cleavage from the solid phase resin. Although this gives the chemo-enzymatic approach an advantage over NCL-based Boc chemistry, the need for an enzyme ligation site introduces some limitations. Furthermore, the chemo-enzymatic approach involves an additional reaction step compared to SPPS with chemical cyclization. Expression of non-native cyclic peptides in plant systems is still in the early stages of development. However, the prospects are promising, and plant expression might take the production of cyclic peptides to an industrial scale in due course. However, more plant and plant cell systems need to be tested and there is a need for a greater understanding of biosynthetic cyclization mechanisms to optimize these expression systems. Bacterial expression systems have been shown to be effective in the expression of the cyclotide MCoTI-I, and several mutants have been generated both by changes to the amino acid sequence [37] and by isotopic labeling with 15N nitrogen [39]. Bacterial expression systems need to be further explored on a wider range of cyclic peptides, but appear to be promising systems for the expression of cyclic peptides. In summary, the three major technologies for cyclic peptide production, that is, isolation, chemical synthesis and biological synthesis, are all complementary and purpose dependent but are still rapidly evolving and promise to facilitate a range of exciting applications of cyclic peptides on the near future.
Conflict of interest DJC is an inventor on patents in the field of cyclotides and other cyclic peptides.
Acknowledgements Work in our laboratory on peptide toxins is supported by grants from the National Health & Medical Research Council (NHMRC; Grant ID nos: APP519734, APP631457 and APP1010552) and the Australian Research Council (DP0984390). DJC is an NHMRC Professorial Research Fellow e20
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(ID no. 569603). LT is a recipient of the Cancer Council Queensland PhD Scholarship.
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