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Cyclotides as a basis for drug design ARTICLE in EXPERT OPINION ON DRUG DISCOVERY · MARCH 2012 Impact Factor: 3.54 · DOI: 10.1517/17460441.2012.661554 · Source: PubMed
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Review
Cyclotides as a basis for drug design
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Queensland on 05/07/13 For personal use only.
David J Craik†, Joakim E Swedberg, Joshua S Mylne & Masa Cemazar 1.
Introduction
The University of Queensland, Institute for Molecular Bioscience, Brisbane, Qld, Australia
2.
Overview of therapeutic potential
3.
Recent progress
4.
Biosynthesis and screening of CCK molecules in vivo
5.
Challenges in working with
Introduction: Cyclotides are plant-made defence proteins with a head-to-tail cyclic backbone combined with a conserved, six cystine knot. They have a range of biological activities, including uterotonic and anti-HIV activity, which have attracted attention to their potential pharmaceutical applications. Furthermore, their unique structures and high stability make them appealing as peptide-based templates for drug design applications. Methods have been developed for their production, including solid phase peptide synthesis as well as recombinant methods. Areas covered: This article reviews the recent literature associated with therapeutic applications of naturally occurring and synthetically modified cyclotides. It includes applications of cyclotides and cyclotide-like molecules as peptide-based drug leads and diagnostic agents. Expert opinion: The ultra-stable cyclotides are promising templates for drug development applications and are currently being assessed for the potential breadth of their applications. For synthetic versions of cyclotides to enter human clinical trials further studies to examine their biopharmaceutical properties and toxicities are required. However, several promising proof-ofconcept studies have established that pharmaceutically relevant bioactive peptide sequences can be grafted into cyclotide frameworks and thereby stabilised, while maintaining biological activity. These studies include examples directed at cancer, cardiovascular disease and infectious diseases. Solid phase peptide synthesis has been the preferred approach for making pharmaceutically modified cyclotides so far, but promising progress is being made in biological approaches to cyclotide production.
cyclotides 6.
Conclusions
7.
Expert opinion
Keywords: circular proteins, cyclic peptides, cyclotides, cystine knot, drug design, kalata B1 Expert Opin. Drug Discov. (2012) 7(3):179-194
1.
Introduction
Cyclotides were first defined as a unique family of proteins in 1999 [1], with the name being a condensation of the terms cyclo and peptide to indicate their major distinguishing characteristic of a head-to tail cyclic peptide backbone. Cyclotides also contain a cystine knot motif made of up of three conserved disulfide bonds. Two of these disulfide bonds and their connecting backbone segments form an embedded ring in the structure that is threaded by the third disulfide bond (Figure 1). Cyclotides are the only known class of peptides that have the combination of a cyclic backbone and a cystine knot, which together are known as a cyclic cystine knot (CCK) motif. This structural motif engenders cyclotides with exceptional stability and this stability is a primary reason for interest in these peptides as pharmaceutical scaffolds. Early indications of the potential applications of cyclotides as therapeutics came from pharmaceutical screening studies of plants [2-4] and from reports of their use in indigenous medicine in Africa [5]. In both cases, the pharmacological activities were discovered before the structures were known and furthermore it
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Cyclotides as a basis for drug design
Article highlights. .
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. .
Cyclotides are ultra-stable peptides that have significant potential as pharmaceutical templates into which bioactive peptide sequences can be grafted. So far, grafting examples have been demonstrated for applications in cancer, cardiovascular disease and inflammation/infectious diseases. Cyclotides can be made using solid phase peptide synthesis or can be expressed recombinantly in plants or micro-organisms. The cyclotide field is a relatively new, but rapidly developing field. No cyclotide-based drug has yet entered clinical trials.
This box summarises key points contained in the article.
was not known at the time that these bioactive molecules would later come to be recognised as members of a large and unique family of cyclic peptides. The prototypic cyclotide is kalata B1, originally discovered in the leaves of the African plant Oldenlandia affinis. This peptide is the active ingredient in a medical tea used by Congolese women to speed up labour and facilitate childbirth [5]. Gran isolated the uterotonic peptide in the early 1970s and partially characterised its amino acid composition [6,7], but the primary sequence and CCK arrangement were not delineated until some 20 years later [8]. Figure 1 shows the structure of kalata B1, the prototypic cyclotide [8,9] highlighting its disulfide connectivity -- CysICysIV, CysII-CysV, CysIII-CysVI -- which forms the cystine knot motif, and the head-to-tail cyclic backbone. These features are thought to be responsible for the exceptional stability of this peptide [10], which withstands boiling and apparent oral ingestion in the indigenous medicinal applications, suggesting resistance to high temperature, digestive enzymes and acidic conditions. A recent study using nuclear magnetic resonance (NMR) relaxation data confirmed the general rigidity of the CCK framework of the cyclotide Momordica cochinchinensis trypsin inhibitor (MCoTI)-I [11,12]. This structural motif is shared by all cyclotides. Examples of these peptides have now been reported in species from the Rubiaceae (coffee), Violaceae (violet), Cucurbitaceae (cucurbit) and Fabaceae (legume) families of plants, with individual plants containing dozens to hundreds of different cyclotides [13]. Cyclotides are gene-encoded [14-20], with the mature cyclotides being biosynthetically excised from larger precursor proteins in a process that involves asparaginyl endopeptidase enzyme activity [21-23]. There are currently more than 200 cyclotides catalogued in Cybase [24], a database dedicated to circular proteins, but it is estimated that the number of naturally occurring cyclotides might approach 50,000 [13]. Associated with this huge potential diversity, cyclotides can be considered as a natural combinatorial template [25] where sequence variations occur
180
in the six backbone loops between the six conserved cysteine residues. Figure 1 highlights this combinatorial diversity and Table 1 gives some example sequences of cyclotides from the three subfamilies of cyclotides: M€obius, bracelet and trypsin inhibitor cyclotides. The diverse range of activities of cyclotides that have been reported in screening studies include anti-HIV [2], uterotonic [26], antimicrobial [27], neurotensin antagonistic [3], antibarnacle [28] and cytotoxic [18,29,30] activities, including haemolytic activity [31]. Although some aspects of their cytotoxic activities are unfavourable from a pharmaceutical development standpoint, their selective toxicity to some cancer cell lines facilitates the possibility of anticancer applications [18,29,30,32,33]. Naturally, it would seem that cyclotides are not produced by plants for the above activities, but rather their native function appears to be to defend plants against insect pests [16,34,35]. They have also been reported to be active against nematodes [36,37] and molluscs [38]. These biocidal functions appear to have a mechanistic basis involving the interactions of cyclotides with membranes in the target organisms. For example, electron microscopy studies have shown that kalata B1 causes membrane disruption in the midgut of caterpillar larvae [39], and various biophysical studies have confirmed the binding of cyclotides to model and/or cell membranes [40-42]. Peptides are generally regarded as excellent drug leads, but in the past have been regarded as poor drugs themselves because of their susceptibility to proteolytic degradation and their generally poor oral bioavailability [43]. The potential pharmaceutical applications of cyclotides are facilitated by their exceptional stability, their amenability to chemical and biological synthesis and their potential to overcome some of these perceived limitations of conventional peptides as drugs. Their pharmaceutical applications can derive from the natural activities of cyclotides, or from their use as templates into which bioactive peptide sequences can be inserted (grafted) so that they might be stabilised. Cyclotides can be chemically synthesised [27,44-46] and the folding pathways of the cystine knot motif have been delineated, thus paving the way for solid phase peptide synthesis routes to the production of pharmaceutically relevant cyclotides [45,47,48]. Recent reviews have covered aspects of cyclotide production [46,49,50] and there have been several recent studies on their biological synthesis. Interestingly, the recent observation [14,15] that cyclotides are naturally produced in a plant from the Fabaceae family, potentially amenable to large-scale cropping, opens the possibility of producing pharmaceutically active cyclotides in transgenic crop plants [14,51]. Some native cyclotide-producing plants make some cyclotides very efficiently, with yields of up to 2 g of cyclotide per kg of plant tissue, and if this can be translated to the production of pharmaceutically modified cyclotides in transgenic plants it would represent probably the most cost-effective way of producing cyclotides.
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Loop 5 41 KSKV 29 KNKV 24 SWPV DPWPV 11 10 KDKV 5 SYPI RGNGY 4 4 SWPI 3 SNKV 3 TWPI 3 SSWPI 3 SKYPL 3 SSKV 2 ENKV GEWKL 2 2 TA FL 2 TWPV GNWGL 2 2 RNRS SCKSDKK 2 NTYRV 2 NKYRV
KRPV SYFP VWPF TNGS TDPI DPFPV SYPV RNRV TDKV NYPI KSSV ETWPV QKGF DELRQ QGKV SLHTLE SIPV KDNL SVANI DTIEKV RSKV SSNV
Loop 4 DDRSDGL QDKV SNRV DPWPI SDKV TSSQ KDKL RNHV SRPV GNYGF NWPV NHHDKV DSSWPI SSMI RNKV KTKV SNNV KDQV SNQV QNKV +6 seqs
S T K I A G Y F V P
26 YRNGIP 18 TRNGLPV 11 YNGIP 9 TRDGLPV 7 YRNGVIP 6 TRNGLPI 5 YKNGSIP GSGSDGGV 4 YGTNGGTIFD 4 3 MRNGIP 3 YLNGTP 3 YKNGTLP 3 TRDGLPT 2 YKNGTFP
146 31 8 7 6 4 2 2
VI V IV II III
VWIP 41 VFIP 35 FGGT 22 VGGT 19 VYIP 19 VYLP 6 FLGT 5 RRDSD 4 TLGT 3 FTGK 3 2 VLIP LLGT 2 VLGT 2 2 FIGK 2 HYIP FKFK 2
Loop 2 VWTG 2 AGGT 2 IFFP HIP TLGK AYFG FGGS FVLP AFGS VAFG VFLG AFIP FTGT TLGE VTGS FFFG
FRGK FEGGN VGGS FTGS FGGR VIERTRAW TTFN IWDKT VFMP AMISF RVIPV VWVP YTFP YVLP FKGK FVLG
AGGR VKGK YVIP FKGW VGNK
I
Loop 1 GES 100 GET 73 21 AES 3 IET PKILKK 3 2 GEG 2 AET GER GDS GDT PKILQR
Loop 6 TKDGSVFN AKDGSIPA YLDGIP THNGLPT YHDKIP YLNSIS TRNGVPI YKNGTIP YRDGVIP YLNGVP YRDGIP YLNGIP YFNGIA YYNGSIP
2 2 2 2 2 2 2 2 2 2 2 2 2 2
SRNGLPV YRNGVP YRKGIP SKNGDIPL TKNGSAIL YRNAP YLDGVP YRAIP YRNGLPV YKNGTP YKDGTLP YNNGLP YFNGSQS +61 seqs
Loop 3 44 NTPG ISSLLG 16 YTPG VTSAIG TVTALLG 10 TVTALIG 8 LT SAIG YTEELG 5 ISAAIG NTEY ISSVVG 5 YTQG 4 ISTLLG YTAG ITTVVG 4 TVTALMG 4 PGA YKPG 4 ITHVPGT ISSAIG ISSVLG 3 FTAVVG 3 NDSS ISGVIG 3 YTVQ ITGIAG LT SAVG 3 ISEMIN TITALLG 3 ISAAVG ISAVLG 2 FNPG 2 ISAIAG YTKG 2 ITAAIG WIPG 2 RIPG NTPY 2 LT STVG FLV 2 ISAALG ISSVIG FTAPLG 2 ISSAVG 2 FLPN FIPG FTGIAG 2 ISYLVG 2 ITGVIG ISSIVG 2 RTVG ISGAIG 2 LTAAIG +33 seqs TVTALVG 2
2 2
2 2 2 2
Figure 1. Structure of the prototypic cyclotide kalata B1, determined by NMR (protein data bank (PDB) ID 1nb1) and a representation of the diversity of peptide sequences seen in the various loops of 208 known, naturally occurring cyclotides extracted from Cybase [24]. The number of times each sequence appears in this 208 (provided it is more than once) is annotated to the right of each sequence in smaller font. The six cysteine residues conserved in all cyclotides are labelled I -- VI, and the loops between adjacent Cys residues are labelled loops 1 -- 6.
Cultured O. affinis plant cells have also been used to produce native cyclotides in excellent yield [52-57], and inteinmediated approaches have been developed to express cyclotides in Escherichia coli [58-60], with yields of up to 0.5 -- 1 mg/l of cultured E. coli. This yield could potentially be increased even more by making use of bacterial fermentors. An in vitro enzyme-mediated cyclisation approach has also been developed in which chemical synthesis is used to assemble a linear cyclotide precursor that is then cyclised via trypsin [61,62]. This enzyme-mediated cyclisation mimics the natural biosynthesis of cyclotides to some extent, although it involves a different enzyme, that is, trypsin instead of asparaginyl endopeptidase.
In summary, naturally occurring cyclotides have a range of biological activities that are potentially exploitable for therapeutic uses, and the fact that cyclotides can be artificially synthesised opens the possibility of making engineered cyclotides with desired pharmaceutical activities. Should any cyclotide be developed as a therapeutic then it could be manufactured synthetically using solid phase peptide chemistry or recombinantly in plants or microorganisms, so production issues are not likely to be limiting in the development of cyclotides. There have been a number of recent reviews on the discovery [25,63], characterisation [64], synthesis [65], structures [66,67] and biology [34,68-70] of cyclotides, but here we focus on
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Expert Opin. Drug Discov. (2012) 7(3)
B B B B B B B B B B B B B B B B B B B B B B M M M M M M M M TI TI
Circulin A Circulin B Circulin C Circulin D Circulin E Circulin F Cyclopsychotride A Cycloviolacin O2 Cycloviolacin O13 Cycloviolacin Y1 Cycloviolacin Y2 Cycloviolacin Y3 Cycloviolacin Y4 Cycloviolacin Y5 Cycloviolin A Cycloviolin B Cycloviolin C Cycloviolin D Kalata B8 Palicourein vhl-1 Vitri A Cycloviolacin O14 Cycloviolacin O15 Cycloviolacin O24 Kalata B1 Kalata B2 Varv A Varv E Varv F MCoTI-I MCoTI-II
C. parvifolia C. parvifolia C. parvifolia C. parvifolia C. parvifolia C. parvifolia P. longipes V. odorata V. odorata V. yedonesis V. yedonesis V. yedonesis V. yedonesis V. yedonesis L. cymosa L. cymosa L. cymosa L. cymosa O. affinis P. condensate V. hederacea V. tricolor V. odorata V. odorata V. odorata O. affinis, V. odorata O. affinis V. arvensis, V. odorata V. arvensis, V. tricolor V. arvensis M. cochinchinensis M. cochinchinensis
Plant species
Bioactivity B, H, V B, H, V V V V V B, C, H, N B, C, F, H C, H H, V H, V H, V H, V H, V V V V V V V V C H H H H, I, U, V H, I C, H C C T T
Sequence G---IP--CGES---CVWIP-CI-S-AAL-G-CSCKN----KVCYR--N GV- -IP--CGES---CVFIP-CI-ST-LL-G-CSCKN- ---KVCYR--N G---IP--CGES---CVFIP-CI-TS-VA-G-CSCKS----KVCYR--N K---IP--CGES---CVWIP-CV-TS-IF-N-CKCEN----KVCYH--D K---IP--CGES---CVWIP-CL-TS-VF-N-CKCEN----KVCYH--D A---IP--CGES---CVWIP-CI-S-AAI-G-CSCKN- ---KVCYR--S---IP--CGES---CVFIP-CTVT--ALLG-CSCKS----KVCYK--N G---IP--CGES---CVWIP-CI-SSAI--G-CSCKS----KVCYR--N G---IP--CGES---CVWIP-CI-S-AAI-G-CSCKS----KVCYR--N GGT-I-FDCGET---CFLGT-CY-T-P---G-CSCGN--Y-GLCYGT-N GGT-I-FDCGES---CFLGT-CY-T-A---G-CSCGN--W-GLCYGT-N GGT-I-FDCGET---CFLGT-CY-T-A---G-CSCGN--W-GLCYGT-N G---VP--CGES---CVFIP-CITGVI---G-CSCSS----NVCY--LN G---IP--CAES---CVWIP-CT-TALV--G-CSCSD----KVCY---N GV--IP--CGES- --CVFIP-CI-SAAI--G-CSCKN----KVCYR--N GT-A----CGES---CYVLP-CF-T-V---G-CTCTS---SQ-CFK--N G---IP--CGES---CVFIP-CL-TTVA--G-CSCKN----KVCYR--N G---FP--CGES---CVFIP-CI-S-AAI-G-CSCKN----KVCYR--N G-S-V-LNCGET---CLLGT-CY-TT----G-CTCNK--Y-RVCTK--D G-D--PTFCGET- --CRVIPVCTYS-AAL-G-CTCDDRS-DGLCKR--N S---I-S-CGES---CAMISFCF-TEVI--G-CSCKN----KVCY--LN G---IP--CGES---CVWIP-CI-TSAI--G-CSCKS----KVCYR--N G-SI-PA-CGES- --CFKGK-CY-T-P---G-CSCSK--Y-PLCAK--N GL-V-P--CGET---CFTGK-CY-T-P---G-CSCS---Y-PICKK--N GL- --PT-CGET---CFGGT-CN-T-P---G-CTCD--PW-PVCTH--N GL---PV-CGET---CVGGT-CN-T-P---G-CTCS---W-PVCTR--D GL---PV-CGET---CFGGT-CN-T-P---G-CSCT---W-PICTR--D GL---PV-CGET---CVGGT-CN-T-P- --G-CSCS---W-PVCTR--N GL---PI-CGET---CVGGT-CN-T-P- --G-CSCS---W-PVCTR--N GV---PI-CGET---CTLGT-CY-T-A- --G-CSCS---W-PVCTR--N GG-V----CPKILQRCRRDSDC----P---GACICRG---NGYCGSGSD GG-V----CPKILKKCRRDSDC----P---GACICRG---NGYCGSGSD
Bioactivity: B: Antibacterial; C: Cytotoxic; F: Marine antifouling; H: Haemolytic; I: Insecticidal; N: Neurotensin antagonist; U: Uterotonic; V: Anti-HIV. €bius; TI: Trypsin inhibitor. Class: B: Bracelet; M: Mo
Class
Cyclotide
Table 1. Representative cyclotide sequences and activities.
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[2,134] [2,134] [134] [134] [134] [134] [3,27] [28,32,33,97] [135] [136] [136] [136] [136] [136] [137] [137] [137] [137] [138] [139] [140] [30] [135] [135] [135] [2,16,26,31] [2,141] [4,142] [4] [4] [143] [143]
Ref.
Cyclotides as a basis for drug design
Craik, Swedberg, Mylne & Cemazar
recent progress in the development of cyclotides as pharmaceuticals.
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2.
Overview of therapeutic potential
As recently described [71], cyclotides have been the subject of a number of patent applications [72-82]. The patent descriptions suggest that there is commercial interest in cyclotides in both the agricultural and pharmaceutical industries [83]. The agriculturally directed patents focus mainly on crop protection applications against plant pests, thereby exploiting what appears to be the natural function of cyclotides. Here we focus on therapeutic applications. Figure 2 provides an overview of the topologies of cyclotides and related cystine knot molecules that have therapeutic potential. The core CCK framework is highlighted in the centre of the diagram, which is divided into two parts, the upper part representing true cyclotide frameworks and the lower part ‘reduced’ cyclotide frameworks. The term ‘reduced’ is used in the sense of meaning that these frameworks lack one or more of the key features of the intact CCK framework. They are included here because, for example, many acyclic cystine knot molecules [84] have been studied for their pharmaceutical potential and it is useful to view cyclotides as being part of a continuum of peptides varying in topological complexity. The arrows emanating from the core CCK framework (panel A in Figure 2) indicate that cyclotides can potentially be modified in a number of ways to explore their therapeutic potential. These modifications include point mutations of individual residues to optimise activity, grafting of a foreign bioactive peptide sequence into a loop region of the cyclotide framework or minimisation of one or more of the cyclotide loops. Point mutagenesis studies (panel B) have been exemplified by a series of recently reported alanine and lysine scans [85,86] in which each individual non-Cys residue of the cyclotide framework was successively replaced with Ala or Lys. These studies revealed a patch of surface-exposed residues that are important for the haemolytic and/or insecticidal activities of cyclotides. Mutating any one of these residues abolishes these toxic activities, thus improving the therapeutic potential of cyclotides. The grafting strategy is highlighted in panel C, with the principle being that one of the six loops of the cyclotide framework can be substituted by a foreign bioactive peptide sequence having a desired therapeutic effect. In the case shown loop 3 is substituted. The concept of minimising the cyclotide framework is included here for completeness (panel D), although so far there have been no studies reported of this type for the CCK framework itself. Acyclic cystine knot frameworks have however been subjected to such engineering studies [84]. The lower half of Figure 2 illustrates that many other therapeutically useful molecules can be conceptually considered to be related to the cyclotide framework. These include analogues in which one or more disulfide bonds
are removed (panels E -- G), analogues in which disulfide bonds are differently configured than in the CCK motif (panel H) and analogues in which the cyclic backbone is broken (panels I -- K), that is, so-called acyclic permutants [87]. This representation is designed to imply topological relationships between the various derivatives and the core CCK framework rather than identity sequences. For example, removal of the II -- V disulfide bond in the CCK framework leads to topology 2E, which corresponds with that of cyclised conotoxin Vc1.1, which was recently shown to be orally active in an animal model of neuropathic pain [88]. Removal of a second disulfide bond leads to a topology equivalent to that seen in sunflower trypsin inhibitor-1 (SFTI-1) [89], shown in panel 2F. This peptide has been used as a framework for the design of drugs directed against kallikrein-related peptidase 4 (KLK4) which is a protease overexpressed in prostate cancer [90]. Finally, removal of the third disulfide bond leads to a conventional cyclic peptide (panel G). Panel H of Figure 2 schematically illustrating the effects of different disulfide connectivity shows a topology corresponding to that of rhesus theta defensin-1 (RTD-1) [91]; in this case the disulfide connectivity is in a ‘laddered’ arrangement [92] rather than the knotted arrangement seen in the CCK framework. RTD1-1 has attracted recent interest for antimicrobial as well anti-HIV activity [93]. Finally, the representative series of acyclic permutants shown in panels I -- K, illustrate that the cyclotide backbone may be broken, leading to molecules that still contain a cystine knot, but have a conventional linear backbone topology [84]. 3.
Recent progress
In this section, recent progress on the use of cyclotides and related frameworks in drug design is described. For convenience, the studies are divided into those involving native cyclotides, grafted cyclotides (i.e., those where one or more loops have been replaced) and reduced cyclotide frameworks involving cyclotide-like molecules that have altered numbers or configurations of disulfide bonds, or have an acyclic peptide backbone. Natural cyclotides Although the natural function of cyclotides appears to be as defence molecules against insect and nematode pests, a number of cyclotides have shown pharmaceutically relevant activities in various screening studies. 3.1
Anti-HIV activity Anti-HIV activity was one of the first activities screened for in cyclotides [2] and this activity has been confirmed in a number of subsequent papers. The structure--activity relationship (SAR) studies have been conducted and have established that a patch of residues co-located on the surface of kalata 3.1.1
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B.
C.
D.
A
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Mutated cyclotide
Grafted cyclotide
II
I
A.
1
Minimised cyclotide
III 2
Cyclotide framework Loop 6 5
‘Cyclotide related’ framework
Less disulfides
E.
Loop 3
VI
4 IV
V
Different connectivity
H.
Non-cyclic
I.
F.
J.
G.
K.
Figure 2. Topologies of a generic cyclotide framework and modified analogues. The structures in the upper half of the figure contain a CCK framework and those in the lower half are related frameworks that have fewer disulfide bonds or lack either the cystine knot or cyclic backbone. (A) Generic cyclotide topology. Cystine residues are labelled I -- VI and the backbone loops are numbered 1 -- 6. (B) Cyclotide point mutant. (C) Grafted cyclotide analogue where loop 3 is replaced by a foreign bioactive peptide sequence. (D) Cyclotide mutant in which one of the loops is reduced in size. (E -- G) Mutants with reduced numbers of disulfide bonds. (H) Cyclic tri-disulfide peptide with a simplified topology relative to the cystine knot of cyclotides. The laddered topology shown corresponds to the q-defensins. (I -- K) Acyclic permutants of cyclotides in which the backbone is broken in various loops.
B1 is important for anti-HIV activity [85]. It has also been shown that the circular backbone is essential for anti-HIV activity [94]. In the most recent study, membrane-binding interactions were found to be important for the antiHIV activity, which appears to result from the ability of the 184
cyclotide kalata B1 to selectively target and disrupt the membrane envelope of HIV particles [95]. Thus, unlike other anti-HIV peptides that inhibit viral entry into a host cell, kalata B1 appears to have direct virucidal activity by targeting the viral envelope which comprises raft-like membranes
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very rich in phosphatidylethanolamine (PE) phospholipids [95]. Biosensor and NMR studies established that kalata B1 makes specific interaction with PE head groups, but is further modulated by specific peptide lipid hydrophobic interactions which are favoured in raft-like domains. Kalata B1 does not specifically target negatively charged phospholipids like many conventional membrane-active peptides. Anticancer activity Cyclotides have cytotoxicity against a number of cancer cell lines derived from different cancer types including myeloma (RPMI-8226), T-cell leukaemia (CCRF-CEM), lung cancer (NCI-H69), lymphoma (U-937GTB) and adenocarcinoma (ACHN) [29]. The potential anticancer activity of cyclotides arises from their ability to selectively target cancer cells over normal cells [29], again with the membrane being the primary target site [96]. The in vitro therapeutic index for primary chronic lymphocytic leukaemia cells is around 10 [29], which is similar to comparable treatments such as doxorubicin. Although a number of studies have shown cytotoxicity in cancer cell lines [30,33], a lack of controls in the form of noncancer cells makes comparisons with current treatments difficult. So far, the promising activity in cell-based assays has not translated into in vivo mouse tumour models where considerable toxicity has been observed [32]. Consequently, the cytotoxicity of cyclotides against cancer cells versus normal cells needs further evaluation.
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3.1.2
Antimicrobial activity The first report of antimicrobial activity in cyclotides was in 1999 [27] and there have been few reports since then. Recent studies suggested some conflicting results with respect to the initial studies [69] and so the true potential of cyclotides as antimicrobial agents is not yet clear. It appears that some cyclotides do exhibit antimicrobial activity under certain tested conditions, but that the activity is not necessarily maintained under high salt conditions that might be encountered in a physiological system. Cycloviolacin O2 appears to be amongst the most active antimicrobial cyclotides [97]. Certainly, the area is worthy of further investigation although at this stage it seems that cyclotides are unlikely to be clinically useful as antimicrobial agents. 3.1.3
Anthelmintic activity Cyclotides have been tested against a variety of livestock and human parasites. They have been shown to have activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematodes in sheep [36]. More recently, they were shown to affect the canine hookworm Ancylostoma caninum, and larvae of Necator americanus, a human hookworm [37]. This is a promising new area worthy of investigation, as the potential exists for cyclotide-producing plants to be used as natural medicines against parasites affecting mainly third world countries. 3.1.4
Overview of natural cyclotides Overall, while naturally occurring cyclotides display a range of pharmaceutically interesting bioactivities, so far none has reached the stage of clinical trials. The major potential challenge here is the low therapeutic index, with for example the ratio of toxic to therapeutic effects in anti-HIV activity being ~10. A significantly higher therapeutic index would be required before cyclotides could be progressed to clinical trials. There is some promise that this might be achieved as alanine scanning studies have revealed residues that can be modified to reduce toxic activities [85]. However, realistically it seems most likely that cyclotides will find their pharmaceutical niche as templates for the grafting of foreign activities rather than being used for their intrinsic biological activities. 3.1.5
Grafted cyclotides The grafting concept has been illustrated in several recent papers. The first was a proof-of-concept study on kalata B1 in which three hydrophobic residues in loop 5 were replaced with hydrophilic residues to demonstrate the tolerance of the cyclotide framework to substitution [98]. Recent therapeutically relevant examples included the grafting of an anti-angiogenic sequence onto the cyclotide kalata B1 [99] for applications in cancer therapy and the development of an inhibitor of a protease from foot-and-mouth disease virus via a grafting study on the MCoTI-II cyclotide framework [100]. These and other examples are described below in more detail. 3.2
Anti-angiogenic activity Tumour growth is dependent on a vascular supply to support the tumour and so anti-angiogenic therapy has recently attracted much interest in the cancer field. Gunasekera et al. [99] recently reported the grafting of bioactive peptide epitopes comprising poly arginine sequences onto the kalata B1 framework as potential anti-angiogenic agents. These compounds acted as stabilized VEGF-A antagonists and, while not yet of sufficient potency for clinical applications, they provide valuable proof-of-concept data. 3.2.1
Angiogenesis Pro-angiogenic agents have significant potential in the treatment of a range of conditions, including wound healing, cardiac ischemia, diabetic retinopathy and rheumatoid arthritis. Chan et al. [101] recently reported grafting of angiogenic peptide sequences derived from extracellular matrix proteins onto the MCoTI-II framework. The novel grafted compounds produced blood vessel growth in a well-established chorioallantoic membrane assay and had higher stability in biological fluids than the linear peptide epitopes. 3.2.2
Inflammatory and infectious diseases The MCoTI-II framework has also been used to design peptides with activity against -tryptase and human leucocyte elastase, which are drug targets implicated in many inflammatory 3.2.3
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diseases [102]. The same framework has also been developed to inhibit the 3C protease of foot and mouth disease virus [100]. This represents the first peptidic inhibitor of this protease and, although only exhibiting micromolar affinity, represents a promising starting point. Comparison of linear and CCK frameworks -- cyclic cell-penetrating peptides
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3.2.4
A recent study examined the role of cyclic backbone in contributing extra stability/bioavailability to the cystine knot framework and it was concluded that a large part of the stability of these peptides is associated with the cystine knot framework itself. The backbone cyclisation is thought to assist mainly by reducing susceptibility to thermal unfolding [103]. Very recently another important role of the cyclic backbone has become apparent, namely that of promoting cell penetration, thereby opening up the opportunity of using the frameworks as carriers to deliver bioactive epitopes to intracellular targets. MCoTI-II was the first disulfide-rich macro-cyclic peptide reported to penetrate cells, as assessed by fixedcell imagining [104]. Subsequent studies confirmed this with live cell imaging [105,106]. More broadly, Cascales et al. [105] recently showed that both M- and T-class cyclotides are able to penetrate cells, as can SFTI-1, leading the authors to define a new class of cell-penetrating peptides called the cyclic cell-penetrating peptides (CCPPs) [105]. Combinatorial libraries and in cell expression The MCoTI-I framework has been used as a valuable framework for expression of modified cyclotides in E. coli. For example, in a recent study residues in loops 1 and 5 were replaced in a proof-of-concept study with various amino acids to determine the influence of mutations on structure and activity [107]. As expected the active site lysine was found to be important for trypsin inhibitor activity. Importantly, the overall folding of these molecules was found to be tolerant to residue substitutions confirming proof-of-concept that the CCK framework is a valuable grafting framework. 3.2.5
Cyclotide-related frameworks We use the term ‘cyclotide-related’ frameworks to refer to peptides lacking one or more of the features of cyclotides, for example, by having fewer disulfide bonds or a simpler disulfide connectivity than the cystine knot. It is important to stress that we are not proposing to rename these other frameworks; rather we are merely aiming to illustrate their topological relationships to cyclotides, which are the focus of this article. 3.3
Sunflower trypsin inhibitor-1 One of the smallest disulfide-containing peptides, SFTI-1 (Figure 3A), is a broad range serine protease inhibitor and one of the most potent known naturally occurring trypsin inhibitors (Ki = 0.1 pM) [89]. SFTI-1, like most protease inhibitors and substrates, binds its target proteases via an 3.3.1
186
extended b-sheet at the active site (Figure 3B) [108]. As a result, preferred linear peptide sequences may be grafted into the SFTI scaffold to target its potent inhibition towards a particular protease. This principle was recently used to redirect SFTI’s inhibitory activity towards KLK4, a protease implicated in prostate cancer progression [109]. The approximate peptide substrate preference of KLK4 was first determined by combinatorial positional scanning peptide library screening [110]. The positional scanning approach outlines the general architecture of the protease substrate binding sites while the combinatorial nature of these libraries means substrate sequences relying on subsite cooperativity cannot be identified [111,112]. Consequently, the substrate specificity of KLK4 was further refined by screening against a noncombinatorial sparse matrix library of peptides [90]. The sparse matrix library contained all possible sequence combinations of residues suggested to be preferred by positional scanning (Figure 3C), and identified a KLK4 substrate (FVQR) that was cleaved at nearly twice the rate of any other. Grafting this sequence into the SFTI scaffold (as FCQR to preserve the bisecting disulfide bond; Figure 3D and E) produced a potent KLK4 inhibitor (Ki = 3.6 ± 0.3 nM) that maintained selectivity over other closely related kallikreins and trypsin [90]. Furthermore, this compound was particularly stable in cell culture (t1/2 = 4 days) and blocked protease-activated receptor signalling by KLK4 in vitro. -Defensins Another class of cyclic peptides that has attracted considerable interest for drug development is the q-defensins (DEFT), which have antimicrobial [113] and antiviral [114] properties. q-Defensins are circular peptides formed by the posttranslational splicing of two nonapeptides (Figure 4A). Although the translation of q-defensins only has been demonstrated in old world monkeys and orangutans [115], six transcribed pseudogenes (DEFTY) containing premature stop codons are present in the human genome [116]. Although they are not translated, five of these would encode identical peptide sequences while DEFT-4Y includes a Gly to Arg substitution (Figure 4B). Knowledge of the pseudogene sequences has allowed for resurrection of the ancestral human qdefensins using solid phase peptide synthesis to produce synthetic q-defensins termed retrocyclins [117]. The structure of one of these, retrocyclin-2, is shown in Figure 4C. As for the non-human q-defensin counterparts, the retrocyclins exhibit antibacterial [118] and antiviral activities [119], and greatly reduce HIV-1 infection of human host cells in vitro [117,120,121]. The mechanism of inhibition of viral replication is thought to rely on lectin-like carbohydrate binding properties and cross-linking of glycoproteins [119,122] and has recently been reviewed [123]. Considering that the human q-defensins have been dormant for around seven million years [116], they are most likely adapted to strains of retroviruses from this period rather than currently prevalent ones. Consequently, screening of 3.3.2
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Expert Opin. Drug Discov. (2012) 7(3)
IIe7
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Figure 3. Engineering of SFTI-1 to target KLK4. (A) Schematic representation of SFTI-showing the primary and secondary structures, with residues conferring key specificity highlighted in dark grey. (B) Ribbon plot of trypsin (light colour; PDB ID 1sfi) SFTI-1 (dark grey) with SFTI-1 at the active site shown in stick format (dark colour). Only interacting side chains are shown labelled with single letter code. Hydrogen bonds are shown with dotted black lines. (C) Sparse matrix library of linear peptide substrates used to screen preference by KLK4 across the active site b-sheet. (D) Incorporation of the preferred substrate FVQR into the SFTI scaffold as FCQR (SFTI-1 with the residue substitutions Arg2 to Phe2, Thr4 to Gln4 and Lys5 to Arg5 (SFTI-FCQR)). (E) Ribbon plot of KLK4 (light colour; PDB ID 2bdg) with SFTI-FCQR at the active site shown in stick format (dark colour). Only the interacting side chains are labelled using single letter code. Hydrogen bonds are shown with dotted black lines.
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Figure 4. Structure of -defensins. (A) Schematic representation of the earliest discovered q-defensins, the RTDs. These cyclic peptides are produced by post-translational splicing (dotted line) of two nonapeptide segments that may be homo- or heterodimers. Sequence diversity generated by different splice variants is highlighted in grey. (B) Schematic representation of the human retrocyclins, with splice sites highlighted with dotted lines and sequence diversity in grey. (C) Ribbon plot of the heterodimer retrocyclin-2 with disulfide bonds shown in ball and stick format (PDB ID 2atg).
peptide libraries containing retrocyclin variants have identified alternative sequences with comparable or enhanced ability (retrocyclin-101) to prevent host cell infection by HIV-1 in vitro [124]. While these findings are encouraging, synthesis of large libraries of cyclic q-defensin analogues is cumbersome, and recent research has focused on producing simplified peptides that mimic the topology of the q-defensins. These peptides consisted of 13 -- 14 amino acids and 1 -- 3 disulfide bonds, but lack the head-to-tail cyclisation of naturally occurring q-defensins to enable complete on-resin solid phase synthesis. Several of these truncated q-defensin analogues showed comparable antiviral activity with the full-length parent compound [125]. Another approach to facilitate cost-effective retrocyclin production could involve recombinant expression in plants. Green fluorescent protein (GFP) with C-terminally fused retrocyclin-101 was recently expressed in tobacco chloroplasts [126]. Although the authors presented no convincing 188
evidence that the peptide was liberated from the fusion protein in vivo or in vitro, interestingly, tobacco plants expressing the GFP--retrocyclin-101 fusion were resistant to infection by tobacco mosaic virus, suggesting a potential avenue for the use of q-defensins in control of plant viral infections.
Biosynthetic approaches for the production of cyclic peptides can potentially be used to generate large libraries in a complementary approach to case-by-case structure-based grafting studies. This approach was recently illustrated by Camarero and collaborators who generated a small library of peptides based on MCoTI-I where the residues in loops 1 -- 5 were replaced with a range of types of amino acids [107]. The study identified residues important for folding and activity of MCoTI-I and illustrated the principles of library generation. A similar biosynthetic approach has also been recently used for the production of an alanine-scan library of SFTI-1 [127]. Once cyclic peptide libraries are expressed, they can be screened in vivo against a range of biomolecular targets within the same cell. Since CCK molecules are extremely stable in vivo they should not degrade, making them particularly suitable for this approach. 5.
Challenges in working with cyclotides
The main challenges associated with working with cyclotides relate to their complex disulfide connectivity and their synthesis. At the level of the research lab these challenges include determining the disulfide connectivity of cyclotides. A range of NMR spectroscopic and chemical methods are now available to de novo delineate disulfide connectivities of cyclotides [48], but with a wealth of comparative spectroscopic data now available the CCK motif in newly synthesized cyclotides can usually be inferred by comparison with chemical shift data with known CCK molecules. Perhaps the largest challenge in commercialising cyclotides as drugs will be manufacturing them cost-effectively. The efforts described above utilising bacterial and plant-based expression systems are likely to be of much benefit here. 6.
Conclusions
Cyclotides have been formally recognised as a protein class since 1999 and, although they have so far only been studied by a few groups, they are beginning to attract more widespread attention. A common feature of cyclotides that is shared with other naturally occurring cyclic proteins [128-132] is their exceptional stability. Cyclotides are unique amongst circular proteins in that they contain both a head-to-tail cyclised backbone and a cystine knot motif, giving them special stability. Their amenability to sequence modification has opened opportunities for their use as stabilising frameworks
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Craik, Swedberg, Mylne & Cemazar
in peptide-based drug design and several proof-of-concept examples have now been published.
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7.
Expert opinion
The key finding in the field so far is that cyclotides are exceptionally stable and offer promising potential as peptidebased drug design templates. Bioactive peptide sequences can be engineered into a cyclotide framework, leading to grafted chimeras that are resistant to proteolysis but maintain biological activity. In principle, pharmaceutically relevant cyclotide frameworks can be manufactured using solid phase peptide synthesis or can be expressed recombinantly in plants or micro-organisms. The main challenges in the field include the need for more detailed pharmacokinetic studies on lead molecules and for the development of higher potency leads. So far, only limited anecdotal information is available about oral bioavailability of cyclotide-like molecules, and leads have not yet reached low nanomolar potency. Nevertheless, we believe that this is an exciting time for the cyclotide field and that it will hopefully just be a matter of time before one or more CCK molecules enter clinical trials. Another need is for the development of approaches to enable efficient and high-yielding expression of pharmaceutical cyclotides in crop plants. Advances being made in elucidating the mechanisms involved in the biosynthesis of cyclotides are likely to fuel improved methods of large-scale manufacture of lead molecules. The recent discovery of cyclotides in a plant from the Fabaceae family [14,51] has significantly expanded
knowledge of the evolution and biosynthesis of cyclotides. In particular, the discovery of a cyclotide domain encoded within an albumin gene indicates that cyclotides have evolved through more than one mechanism. Interestingly, SFTI-1, which only contains a single disulfide bond instead of the knotted connectivity of cyclotides, is also encoded within an albumin gene [133], albeit a very different albumin and precursor gene to that of the Fabaceae cyclotides. Despite the differences in genes encoding cyclotides and SFTI-1, both appear to involve an asparaginyl endopeptidase for processing into the mature peptides. Elucidating the mechanisms involved in the biosynthesis of cyclotides and SFTI-1 will open up the possibilities for generating these peptides and pharmaceutically relevant analogues in plants, thereby potentially providing low cost routes to manufacture.
Acknowledgements The authors thank D Wilson and Q Kaas for assistance with figures and AC Conibear for helpful comments on the manuscript.
Declaration of interest Work in the authors’ laboratory on cyclotides is funded by grants from the Australian Research Council (ARC) (DP0984955) and the National Health and Medical Research Council (Australia) (1009267). DJ Craik is a NHMRC Professorial Fellow (569603) and JS Mylne is an Australian Research Council QEII Fellow (DP0879133).
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Affiliation
David J Craik†, Joakim E Swedberg, Joshua S Mylne & Masa Cemazar † Author for correspondence The University of Queensland, Institute for Molecular Bioscience, Brisbane, 4072, Qld, Australia Tel: +61 7 3346 2019; E-mail:
[email protected]
