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Cyclotides as drug design scaffolds
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ScienceDirect Cyclotides as drug design scaffolds David J Craik and Junqiao Du Cyclotides are ultra-stable peptides derived from plants. They are around 30 amino acids in size and are characterized by their head-to-tail cyclic backbone and cystine knot. Their exceptional stability and tolerance to sequence substitutions has led to their use as frameworks in drug design. This article describes recent developments in this field, particularly developments over the last two years relating to the grafting of bioactive peptide sequences into the cyclic cystine knot framework of cyclotides to stabilize the sequences. Grafted cyclotides have now been developed that interact with protein or enzyme targets, both extracellular and intracellular, as well as with cell surface receptors and membranes. Address Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia Corresponding author: Craik, David J ([email protected])
its presence in a plant extract used as a uterotonic medicine in the Congo region of Africa in the 1960s. In that application women boiled the above-ground parts of Oldenlandia affinis plants to make a tea which was ingested during labour to accelerate childbirth [7]. This application implies two things: (i) that the peptide survives boiling; and (ii) it survives the digestive tract and is absorbed sufficiently to exert its uterotonic effect. The structure of kalata B1 was first determined in 1995 [8], revealing the CCK motif, which helped to explain the exceptional stability of kalata B1 and its associated favourable pharmaceutical properties. A wide range of other cyclotides have been discovered since in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae and Fabaceae plant families [2] and cyclotide sequences are documented and regularly updated in the publicly accessible database CyBase [9]. It is conservatively estimated that the cyclotide family might comprise more than 50 000 members [10].
Current Opinion in Chemical Biology 2017, 38:8–16 This review comes from a themed issue on Next generation therapeutics Edited by David Craik and Sonia Troeira Henriques
http://dx.doi.org/10.1016/j.cbpa.2017.01.018 1367-5931/ã 2017 Elsevier Ltd. All rights reserved.
Introduction Cyclotides [1,2] are disulfide-rich peptides from plants that have a head-to-tail cyclic backbone and cystine knot arrangement of three conserved disulfide bonds, which are linked CysI-CysIV, CysII-CysV and CysIII-CysVI. The combination of the cystine knot motif and cyclic backbone is referred to as a cyclic cystine knot (CCK) and is responsible for the exceptional stability of cyclotides. Their natural function is thought to be plant defence molecules, as they are expressed in a wide range of plant tissues and have activities against insects [3], nematodes [4], and molluscs [5], driven principally by their ability to form pores in membranes [6]. However, the exceptional stability of cyclotides has generated additional interest in their use in protein engineering and drug design applications. Figure 1 shows the sequence and structure of the prototypical cyclotide kalata B1, originally discovered based on Current Opinion in Chemical Biology 2017, 38:8–16
Several other properties, aside from their exceptional stability, have driven pharmaceutical interest in cyclotides. These include their tolerance to substitutions in their backbone loops, their potential for oral bioactivity [11], and most recently, the finding that some cyclotides are able to penetrate cells and thereby potentially interact with intracellular pharmaceutical targets [12,13]. There are currently 380 published papers primarily focused on cyclotides and much of this literature has been extensively reviewed over the last decade. Thus, here we do not attempt to discuss their discovery, natural functions, structural characterisation, or biosynthesis, which have been extensively reviewed elsewhere. (Table 1 provides a list of selected key reviews [14,15,16,17–23,24] from the last three years that cover these and other background topics). Rather, our focus here is solely on the use of cyclotides as frameworks in drug design [25–40]. These drug design applications have been underpinned by a number of fundamental studies on the synthesis and structural characterisation of cyclotides over the last decade, as schematically illustrated in Figure 1. Furthermore, studies on the cell penetration properties of cyclotides over the last few years have contributed to the possibility of using engineered cyclotides against intracellular pharmaceutical targets. The development of chemical methods for the synthesis of cyclotides has been centrally important to their applications as drug design scaffolds. Specifically, solid phase peptide synthesis and native chemical ligation [41,42,43–46] is routinely used to assemble the cyclic www.sciencedirect.com
Cyclotides as drug design scaffolds Craik and Du 9
Figure 1
Kalata B1
(a)
II
I
IV
III
VI
V
loop
2
lo
Cyclotide (CCK) scaffold
(b)
op
5
loop 6 V
loo VI p4
II III
lo
I
IV
1 op
loop 3
(c)
Synthesis
Structure
(31 papers)
(61 papers)
Information on targets and epitopes
Cell penetration (7 papers)
Intracellular targeting
Grafting (25 of 52 papers) Reviews
Framework optimisation
(16 papers)
(11 papers)
DRU NS G DES IGN APPLICATIO
Drug leads Current Opinion in Chemical Biology
Structure of the prototypic cyclotide kalata B1 and schematic overview of drug design applications. (a) Peptide sequence in one-letter amino acid code showing the disulfide connectivities. (b) Three dimensional structure (PDBID: 1NB1) showing the cystine knot arrangement of the three conserved disulfide bonds and the labelling of backbone loops between successive Cys residues. Cyclotides range in size form 28–37 amino acids, with the variation reflecting different loop lengths. It is estimated that the cyclotide family will comprise approximately 50 000 members [10]. (c) Schematic illustration of how recent fundamental studies on the synthesis, structures, and (for intracellular targets) cell penetration, have underpinned applications in drug design. The numbers of papers referred to in each box include papers published from 2006 onwards on the indicated topic.
backbone of cyclotides, which are well suited to this approach due to their multiple Cys residues. Recombinant methods have also been utilised to make cyclotides [47], as have chemoenzymatic cyclisation approaches [48,49]. These technologies typically are used to make engineered or grafted cyclotides in which one of the native loop sequences is replaced by a loop having a desired bioactivity. Structural studies have been important in the design and validation stages of these applications. Such structural studies of cyclotides have mainly involved NMR methods and there are currently 49 entries for cyclotides or related molecules in the PDB, 42 of which were determined via NMR and seven with X-ray crystallography. www.sciencedirect.com
Over the last decade there have been 52 papers in the literature focusing primarily on drug design applications of cyclotides, including 25 ‘grafting’ papers [11,12 ,13,50–70,71] where the aim is to introduce a desired activity into a cyclotide framework, 11 papers concerning the synthesis/optimisation of the CCK framework [72– 78,79,80–82] and 16 reviews on drug design applications of cyclotides [25–40]. This information is summarised in Figure 1, which provides a schematic illustration of how inputs from fundamental studies of cyclotide synthesis, structure and cell penetration, combined with inputs from the literature on the sequences of bioactive peptides have underpinned applications used to design grafted cyclotides to hit specific targets. Current Opinion in Chemical Biology 2017, 38:8–16
10 Next generation therapeutics
Table 1 Selected recent reviews on cyclotides Title
Topic General review Historical accounts Discovery Sequencing Structure Bioactivity Synthesis Biosynthesis Membrane binding Membrane binding Biotechnological applications Drug design applications
Ref.
Chemistry and biology of cyclotides: circular plant peptides outside the box Joseph Rudinger memorial lecture: discovery and applications of cyclotides Discovery, structure, function, and applications of cyclotides: circular proteins from plants Primary structural analysis of cyclotides Structural studies of cyclotides Circling the enemy: cyclic proteins in plant defence Chemical and biological production of cyclotides Cyclotide biosynthesis Importance of the cell membrane on the mechanism of action of cyclotides The increasing role of phosphatidylethanolamine as a lipid receptor in the action of host defence peptides Cyclotides in a biotechnological context: opportunities and challenges There have been sixteen reviews on this topic in since 2006, with most focussing in the use of the CCK as a scaffold, including three [38–40] post 2014 reviews
Figure 2 summarises the grafting concept and highlights that a variety of target receptor types and cellular locations of targets have been explored, ranging from extracellular enzymes to cell surface receptors, membranes, and even intracellular targets, including kinases [13] or protein:protein interactions [12]. In all of these applications just two subfamilies of cyclotide frameworks have been used. Cyclotides are actually divided into three subfamilies, the Mo¨bius, bracelet, and trypsin inhibitor subfamilies. Mo¨bius and bracelet cyclotides are most similar to each other, differing mainly by the presence or absence of a cis-Pro peptide bond in loop 5 of the backbone sequence whereas the trypsin inhibitor cyclotides have quite different sequences. Because bracelet cyclotides are more difficult to correctly fold, almost all grafting studies have focused on Mo¨bius or trypsin inhibitor scaffolds. The fact that bracelet cyclotides cannot practically be used as grafting frameworks is not a major limitation, given the availability of the two other complementary scaffolds, but the development of efficient folding pathways for bracelet cyclotides in future would be valuable to evaluate their potential applications. Recent studies have attempted to unravel the reasons for the difficult folding of bracelet cyclotides by examining chimeric bracelet/Mo¨bius cyclotides [44–46] and a range of other mutated cyclotides. Table 2 provides an analysis of the grafted cyclotides that have been reported in the literature to date, showing the epitope sizes and grafting locations (i.e., loops) used for the two utilised CCK framework subfamilies (Mo¨bius and trypsin inhibitor). The former subfamily framework tends to be used for extracellular targets and for cases when membrane binding might be advantageous, and the latter for intracellular targets or those involving enzyme inhibition. The table emphasises the vast range of applications of grafted cyclotides, from cancer to metabolic disease, pain and multiple sclerosis. It also emphasises the versatility of the CCK framework and its tolerance to loop Current Opinion in Chemical Biology 2017, 38:8–16
[18] [16] [24] [19] [20] [14] [22] [17] [15] [23] [21] [25–40]
substitutions of vastly varying content and size. For example, each and every one of the six backbone loops of the CCK framework has been used to incorporate bioactive epitopes, and the sizes of inserted epitopes have ranged from 1 to 21 amino acids (Table 2). We note that the grafts involving just single residue substitutions can equivalently be regarded as point mutations, and that such mutations have, in some cases, been used to optimize properties of the framework rather than introducing a target bioactivity per se. For example, single Lys substitutions in kalata B1 were used to increase membrane targeting for enhancing the nematocidal activities of cyclotides [83]. Nevertheless, in Table 2 we classify selected examples of these single-residue substitutions as examples of ‘grafting’ to highlight the generality and limits of the grafting approach. It is clear from Table 2 that most grafting studies have focused on single loop grafts, with typical epitope sizes of 6–8 amino acids. Loop 6 has been the most commonly grafted loop and loop 6 grafted cyclotides typically fold well for either Mobius or trypsin inhibitor frameworks. Loop 1 has so far only been used as a grafting site for trypsin inhibitor cyclotides, mainly owing to the fact that this is the reactive site loop of MCoTI peptides and thus naturally accommodates protease inhibitory activity, which can be tweaked for quite a wide range of different proteases. For example, loop 1 grafts have been used to make inhibitors of matriptase, foot-and-mouth-disease virus 3C protease, b-tryptase, Bcr-Abl kinase and FXIIa (Table 2). Other trends apparent from Table 2 are that, so far, loops 2 and 3 have been relatively underrepresented in grafting studies, and that it is possible to include unnatural chirality in grafts. For example, the development of agonists of the MCR4 receptor as potential treatments for obesity involved insertion of the epitope GHfRWG into loop 6 of kalata B1 (‘f’ denotes D-Phe). It is also worth noting that this example illustrates that
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Cyclotides as drug design scaffolds Craik and Du 11
agonists, as well as antagonists of receptors, can be developed using cyclotide grafting technology. Although most grafting studies have involved single loop substitutions, the simultaneous grafting of two or more of the six CCK loops is possible, and has been used in cases where the target epitope is particularly large, or in cases where dual targeting or dual functionality is desired [68 ]. An example of the former is the grafting of sequences from myelin oligodendrocyte glycoprotein (MOG) into kalata B1 for the development of drug leads for multiple sclerosis [61]. In that case, a 22-amino acid epitope spanned both loops 5 and 6 of kalata B1, with a Cys residue incorporated into the grafted epitope in a position that allowed the native disulfide bond to be retained in the CCK scaffold. An example of dual targeting was recently reported for an anti-angiogenic cyclotide, with Figure 2
(a) cell surface
(d) intracellular
(c) extracellular
(b) membrane
Grafting
Grafting Bioactive epitope(s)
kalata B1
MCoTI-II
Stable cylotide frameworks
the epitopes YwKV (‘w’ denotes D-Trp) and YHLNQPF grafted into loops 5 and 6, respectively, of MCoTI-II [68]. In our opinion, such dual targeting will be an area of increasing application in future, and represents a particular advantage of the cyclotide framework over some other peptide-based modalities. More complex grafting approaches involving modification of the cystine knot core or cyclic backbone are also possible in combination with grafting. For example, one study to develop a NS2B-NS3 Dengue protease inhibitor involved the use of a two-disulfide kalata B1 variant in which the CysII-CysV disulfide bond was removed via Cys residue substitutions and grafts were effectively inserted into loops 2 and 5, along with single point mutations in other loops. The authors of that study reported the presence of multiple isomers of the resultant two-disulfide product, and the exact structure of the final product was unclear, but at least the principles of simultaneously using several different engineering approaches is illustrated by this study [54]. Another example involved the use of a hybrid cystine knot peptide that combined two different inhibitory sequences and was cyclised via hydrazone formation. This engineered cyclotide had high inhibitory activity against trypsin (Ki = 0.1 nM) and tryptase (Ki = 1 nM) [55]. The size of cyclotides (around 30 amino acids, with a MW of 3000 Da) fills a gap between traditional small molecule drugs (MW <500 Da) and larger biologics (MW >5000 Da) such as antibodies [84] and to some extent this gives them some advantages over both: being bigger than small molecules they are more suited for blocking protein: protein interactions, and being smaller than antibodies they have better cell penetrating properties. Thus, cyclotides potentially can access targets that are inaccessible to either small molecules or antibodies. Of particular current interest is the use of cyclotides as carriers to deliver bioactive cargoes inside cells. The first published example exemplifying this principle involved the use of a grafted trypsin inhibitor cyclotide (MCoTI-I) to modulate the p53 HDM2 intracellular protein-protein interaction to reduce tumour growth in a mouse xenograft model [12]. Another recent example was the use of grafted MCoTI-II to targeting the intracellular SET protein implicated in leukaemia [13].
Current Opinion in Chemical Biology
Schematic illustration of the grafting process to highlight the diverse range of targets accessible to cyclotides. These targets range from: (a) cell surface receptors [51], to (b) cell membranes [15], to (c) extracellular targets (e.g., FMDV 3C protease [52]), to (d) intracellular targets [12,13]. Two types of cyclotide scaffolds are shown: kalata B1 as a representative of the Mo¨bius subfamily, and MCoTI-II as a representative of the trypsin inhibitor subfamily. The grafting process involves insertion of a bioactive epitope into one of these stable CCK frameworks. In the examples shown, loop 6 has been used to incorporate a bioactive helix. www.sciencedirect.com
Finally, since the natural function of cyclotides is as host defence agents, with a common mechanism of action involving membrane binding and disruption, there is an opportunity to use controlled membrane targeting of cyclotides for peptide epitope delivery. Recent studies have shown that the membrane binding of kalata B1 is mediated by interactions with phosphatidylethanolamine [15] and thus lipid selectivity might in principle be used to selectively target cyclotides to cells with particular lipid compositions on their surface. Supporting the proposal of Current Opinion in Chemical Biology 2017, 38:8–16
Summary of published grafting studies using CCK framework as a drug design scaffold. Biological activity Mo¨bius subfamily
Grafted sequence
5
VEGF-A antagonist
2 3 3 5 5 6 2&5
DK KNK RRKRRR RRKRRR NTRRKRRRG RRKRRR RRKRRRV RRKRRR SEESRRG&RRSR c
6 6 6 6 6 6 5&6
GHFRWG GHfRWGe HFRW HfRWe KRPPGFSPL QIPGLGPL KPLR& KAPRMVR i
Experimental autoimmune encephalomyelitis
5 5 6 5&6 4 2
RSPFSRV 6 others 6 others 4 others K K
HIV gp120 inhibitor
5&6
Sl&GSFLRFLTKG
Dengue NS2B-NS3 protease inhibitor Melanocortin 4 receptor (MC4R) agonist
Bradykinin B1 receptor antagonist Neuropilin-1/-2 antagonist
Immunomodulation (Experimental autoimmune encephalomyelitis, EAE, mouse model)
Trypsin inhibitor subfamily
Loop
Proof-of-concept studya
CXCR4 antagonist, HIV-1 cell entry blocker Hdm2/HdmX antagonist
FMDV 3C protease inhibitor
b-tryptase inhibitor b-tryptase and HLE inhibitor
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Angiogenic Trypsin and matriptase inhibitor CTLA-4 Synuclein-induced cytotoxicity
5&6 6 6 6 6 6 6 6 1 1 1 3, 5 & 6 3, 5 & 6 1 1 6 6 6 6 6 1, 3 & 6 1, 3 & 6 6
l
S &GSFLTGQGSF YRXCRGpRRBCYXKn 7 others GASRAPTSFAEYWNLLSA GASKAPTSFAEYWNLLSA GASKAPTSAAEYWNLLSA GASRAPTSAAEYWNLLSA GASRAPTSFAEYZNLLSAp Q AKQ F+3 others r LAG, GPNGF&AKKVHseZs LAG, GPNGF&SHseZDG s V 6 others SDGGl 7 others SIKVAV SVVYGLR R 15 others KYSHVP, K&PR 9 others SLATWAVG
Potency/dose
Application
–
Ref.
Confirm plasticity of CCK scaffold Anti-angiogenesis with the potential to reduce blood vessel growth in tumours.
[50]
1.39 0.35d 3.03 0.75 d 580 f
Dengue Fever
[54]
Obesity
[58]
49%g, 42%h 38%g, 28% h 100i f
Chronic pain & inflammatory pain Inhibit endothelial cell migration, angiogenesis, lymphangiogenesis Multiple sclerosis
[11]
12 b
28.116.6j
20 k
1067 m
[51]
[59]
[61]
Multiple sclerosis, T-cellmediated disorders
[71]
Anti-HIV
[66]
Anticancer and anti HIV-1
[57]
Inhibit tumour growth via modulation of intracellular protein-protein interaction
[12]
Foot-and-mouth disease
[52]
Allergic asthma and inflammation disorders Inflammatory disease
[55]
m
959 20f,o, 2f,o 2.3 0.1 , 9.7 0.9 q
2.6 0.4 q 41 25d 56 35d > 100 d 0.001d 4d >50d t, 0.021d 0.009
d t
q
t
, 0.0025
14.6u 13.9 u 0.00001d,v, 0.00029d,v 3.7
w
[53]
d t
Cardiovascular disease and/or wound healing Anticancer Immunotherapy of cancer and other diseases Explore the feasibility of phenotypic screening of bioactive cyclotides
[56] [60] [65] [64]
12 Next generation therapeutics
Current Opinion in Chemical Biology 2017, 38:8–16
Table 2
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Table 2 (Continued ) Biological activity
Loop
Grafted sequence
Potency/dose
EAIYAAPK&GEAIYAAPFAR 4 others 9 others 2 others GViTRIR x GNKRTRG GNKRTRG GNKRTRG GASKAPASXLRKLXKRLLRDAz GASKAPASXLRKLXKRLL z EIVYRXA
1.3 0.1
Angiotensin (1–7) receptor
1&6 1 6 1&6 6 1 2 6 6 6 6
Anti-angiogenic
5&6
1B,f
FXIIa and FXa inhibitors
1 1 1 1
YwKV&YHLNQPFB 6 others FR&Q SR&Q FRW&Q FRW&KQ
BCR-ABL kinase inhibitor
Cell migration inhibitor LyP1 marker for tumour lymphatics SET antagonist
& & & &
6 6 6 6
b
>128b,y >128/n.d. b,y >128 b,y 2.9 0.14b,z 5.0 0.15b,z
0.1C, 0.46C 2.16C, 96C 0.51C, >100C 0.49C, >100 C
Application
Ref.
Chronic myeloid leukaemia
[63]
Anti-angiogenesis Anti-tumour and anticancer
[62] [69]
Anticancer
[13]
Lung cancer or myocardial infarction Anticancer
[67]
Thrombosis, cardiovascular diseases
[68] [70]
a
Proof-of-concept study to demonstrate that loops are tolerant to modification. IC50, half maximal inhibitory concentration (mM). C9, C21 are substituted by E and R (bold), respectively; There are also mutations E7S, T8E, T20R and N29R in loops1, 4 and 6. The two isomers of grafted cyclotides have Kis of 1.39 0.35 mM (isomer B) and 3.03 0.75 mM (isomer C), respectively. d Ki, inhibition constant (mM). e Residue f refers to D-phenylalanine. f EC50, half maximal effective concentration (nM). g Inhibition of writhing after intraperitoneal injection (i.p. 1 mg/kg). h Inhibition of writhing under oral administration (p.o. 10 mg/kg). i There are mutations of V10 M in loop2 and N15R, P17F in loop3, the EC50 was tested on HUVEC cells. j Cumulative clinical score using experimental autoimmune encephalomyelitis mouse model was significantly lower than the cumulative score of control group. k Oral administration (mg/kg). l Truncated residue/s. m The binding affinity was determined by measuring the non-covalent interaction energy, which is a combination of electrostatic and VDW interaction energies (kJ/mol). n Single letter codes B, X, and p represent the amino acids 2-naphthylalanine, citruline, and D-proline, respectively. o A potent CXCR4 antagonist with EC50 20 nM and an efficient HIV-1 cell-entry blocker with EC50 2 nM. p Z = 6-chlorotryptophan. q Binding affinity of grafted cyclotides to recombinant Hdm2 and HdmX was measured by fluorescence polarisation anisotropy. Cyclotide MCo-PMI-K37R displayed strong affinity for Hdm2 (KD = 2.3 0.1 nM) and HdmX (KD = 9.7 0.9 nM); MCo-PMI-6ClW had affinity of Hdm2 (KD = 2.6 0.4 nM). r Another three cyclotides have mutations K10R, K10V, K10A. s Hse, homoserine; Z, N-terminal glyoxylyl residue. t MCoTI-II[K10V] displayed inhibitory activity of Ki >50 mM (tryptase) and Ki 0.021 mM (HLE); MCoTI-II[SDGG truncated] had inhibitory activity of Ki 0.009 mM (tryptase), and Ki 0.0026 mM (trypsin). u The lowest concentration with significant difference compared to control (mM). v MCoTI-II[V3R] displayed inhibition of Ki 0.00001 mM (trypsin) and Ki 0.00029 mM (matriptase). w Binding constant was estimated using ELISA(mM). x Letter i = D-Isoleucine. y Cytotoxicity of grafted cyclotide in loop 1and loop 6 against normal and cancer cell lines were same as IC50>128 mM (MDA-MB-435S, MDA-MB-231, MM96L, HFF1), the IC50 of grafted cyclotide in loop 2 was >128 mM in MDA-MB-435S and not determined in other three cell lines. z Letter X represents amino-isobutyric acid. The IC50 was tested against K562 cells. A Residue X represents L-2,3-diaminopropionic acid. B Residue w represents D-tryptophan, the EC50 was tested on HUVEC cells. C Inhibition constants of each grafted cyclotide for FXIIa and FXa are Ki 0.11 mM and 0.46 mM, 2.16 mM and 96 mM, 0.51 mM and >100 mM, 0.49 mM and >100 mM, respectively. b c
Cyclotides as drug design scaffolds Craik and Du 13
Current Opinion in Chemical Biology 2017, 38:8–16
14 Next generation therapeutics
selectivity in membrane targeting, another study showed that cyclotides from different subfamilies (Mo¨bius and bracelet) differed in their orientation when binding to membranes [85]. It has also been reported that cycloviolacin O2 (a bracelet cyclotide) has stronger cytotoxicity against various cancer cell lines than kalata B1 or kalata B2 (Mo¨bius subfamily members) and was more potent towards anionic DOPC/DOPA liposomes than zwitterionic DOPC, suggesting a degree of cell specificity related to the number of positive charged residues in the cyclotide framework [86].
Conclusions Cyclotides are now a widely-studied family of plant proteins and are gaining acceptance as potential drug design scaffolds. No cyclotides have yet reached human clinical trials but, in our opinion, there is a good possibility that this may occur in the not too distant future. The biggest challenge in the field at the moment is increasing the oral bioavailability of peptide-based leads. Although there are several reported cases where cyclotides have oral activity, there is limited literature on the actual oral bioavailability and so more quantitative studies reporting such biopharmaceutical parameters are expected over coming years. This information will make a valuable addition to the wealth of information currently available on the stability and pharmaceutical ‘graftability’ of cyclotides.
Acknowledgements Work on cyclotides in our laboratory is supported by the Australian Research Council (DP150100443) and the NHMRC, Australia (APP1084604). DJC is an ARC Australian Laureate Fellow (FL150100146).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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2.
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9.
Mulvenna JP, Wang C, Craik DJ: CyBase: a database of cyclic protein sequence and structure. Nucleic Acids Res 2006, 34: D192-D194.
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24. Weidmann J, Craik DJ: Discovery, structure, function, and applications of cyclotides: circular proteins from plants. J Exp Bot 2016, 67:4801-4812. The most recent review giving an overview on the discovery and applications of cyclotides. www.sciencedirect.com
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maar M, Wang CK, Daly NL: The cyclotide family of 26. Craik DJ, Ce circular miniproteins: nature’s combinatorial peptide template. Biopolymers: Pept Sci 2006, 84:250-266.
47. Aboye TL, Camarero JA: Biological synthesis of circular polypeptides. J Biol Chem 2012, 287:27026-27032.
27. Craik DJ, Clark RJ, Daly NL: Potential therapeutic applications of the cyclotides and related cystine knot mini-proteins. Expert Opin Investig Drugs 2007, 16:595-604. 28. Kolmar H: Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins. FEBS J 2008, 275:2684-2690. 29. Daly NL, Craik DJ: Design and therapeutic applications of cyclotides. Future Med Chem 2009, 1:1613-1622. 30. Craik DJ, Mylne JS, Daly NL: Cyclotides: macrocyclic peptides with applications in drug design and agriculture. Cell Mol Life Sci 2010, 67:9-16. 31. Jagadish K, Camarero JA: Cyclotides, a promising molecular scaffold for peptide-based therapeutics. Biopolymers: Pept Sci 2010, 94:611-616. 32. Daly NL, Craik DJ: Bioactive cystine knot proteins. Curr Opin Chem Biol 2011, 15:362-368. 33. Gracy J, Chiche L: Structure and modeling of knottins, a promising molecular scaffold for drug discovery. Curr Pharm Des 2011, 17:4337-4350. 34. Smith AB, Daly NL, Craik DJ: Cyclotides: a patent review. Expert Opin Ther Pat 2011, 21:1657-1672. 35. Craik DJ, Swedberg JE, Mylne JS, Cemaar M: Cyclotides as a basis for drug design. Expert Opin Drug Discov 2012, 7:179-194. 36. Poth AG, Chan LY, Craik DJ: Cyclotides as grafting frameworks for protein engineering and drug design applications. Biopolymers: Pept Sci 2013, 100:480-491. 37. Schroeder CI, Swedberg JE, Craik DJ: Recent progress towards pharmaceutical applications of disulfide-rich cyclic peptides. Curr Protein Pept Sci 2013, 14:532-542. 38. Ackerman SE, Currier NV, Bergen JM, Cochran JR: Cystine-knot peptides: emerging tools for cancer imaging and therapy. Expert Rev Proteomics 2014, 11:561-572. 39. Thell K, Hellinger R, Schabbauer G, Gruber CW: Immunosuppressive peptides and their therapeutic applications. Drug Discov Today 2014, 19:645-653. 40. Mollica A, Costante R, Stefanucci A, Novellino E: Cyclotides: a natural combinatorial peptide library or a bioactive sequence player? J Enzyme Inhib Med Chem 2015, 30:575-580.
48. Nguyen GKT, Wang SJ, Qiu YB, Hemu X, Lian YL, Tam JP: Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat Chem Biol 2014, 10:732-738. Paper reporting the isolation of the enzyme butelase-1 with broad utility for cyclising peptides. 49. Harris KS, Durek T, Kaas Q, Poth AG, Gilding EK, Conlan BF, Saska I, Daly NL, van der Weerden NL, Craik DJ et al.: Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat Commun 2015, 6:10199. Paper reporting the recombinant production of the enzyme OaAEP1b with broad utility for cyclising peptides. 50. Clark RJ, Daly NL, Craik DJ: Structural plasticity of the cyclic-cystine-knot framework: implications for biological activity and drug design. Biochem J 2006, 394:85-93. 51. Gunasekera S, Foley FM, Clark RJ, Sando L, Fabri LJ, Craik DJ, Daly NL: Engineering stabilized vascular endothelial growth factor-A antagonists: synthesis, structural characterization, and bioactivity of grafted analogues of cyclotides. J Med Chem 2008, 51:7697-7704. 52. Thongyoo P, Roque-Rosell N, Leatherbarrow RJ, Tate EW: Chemical and biomimetic total syntheses of natural and engineered MCoTI cyclotides. Org Biomol Chem 2008, 6:1462-1470. 53. Thongyoo P, Bonomelli C, Leatherbarrow RJ, Tate EW: Potent inhibitors of b-tryptase and human leukocyte elastase based on the MCoTI-II scaffold. J Med Chem 2009, 52:6197-6200. 54. Gao Y, Cui T, Lam Y: Synthesis and disulfide bond connectivityactivity studies of a kalata B1-inspired cyclopeptide against dengue NS2B-NS3 protease. Bioorg Med Chem 2010, 18:1331-1336. 55. Sommerhoff CP, Avrutina O, Schmoldt HU, Gabrijelcic-Geiger D, Diederichsen U, Kolmar H: Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase b. J Mol Biol 2010, 395:167-175. 56. Chan LY, Gunasekera S, Henriques ST, Worth NF, Le SJ, Clark RJ, Campbell JH, Craik DJ, Daly NL: Engineering pro-angiogenic peptides using stable disulfide-rich cyclic scaffolds. Blood 2011, 118:6709-6717. 57. Aboye TL, Ha H, Majumder S, Christ F, Debyser Z, Shekhtman A, Neamati N, Camarero JA: Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity. J Med Chem 2012, 55:10729-10734.
41. Dawson PE, Muir TW, Clark-Lewis I, Kent SB: Synthesis of proteins by native chemical ligation. Science 1994, 266:776-779.
58. Eliasen R, Daly NL, Wulff BS, Andresen TL, Conde-Frieboes KW, Craik DJ: Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1. J Biol Chem 2012, 287:40493-40501.
42. Aboye T, Kuang Y, Neamati N, Camarero JA: Rapid parallel synthesis of bioactive folded cyclotides by using a tea-bag approach. ChemBioChem 2015, 16:827-833. Paper reporting rapid parallel synthesis of a small library of bioactive cyclotides.
59. Getz JA, Cheneval O, Craik DJ, Daugherty PS: Design of a cyclotide antagonist of neuropilin-1 and -2 that potently inhibits endothelial cell migration. ACS Chem Biol 2013, 8:1147-1154.
43. Akcan M, Craik DJ: Synthesis of cyclic disulfide-rich peptides. In Peptide Synthesis and Applications. Edited by Jensen KJ, Tofteng Shelton P, Pedersen SL. Humana Press; 2013:89-101. Methods in Molecular Biology, vol. 1047.
60. Quimbar P, Malik U, Sommerhoff CP, Kaas Q, Chan LY, Huang Y-H, Grundhuber M, Dunse K, Craik DJ, Anderson MA et al.: High-affinity cyclic peptide matriptase inhibitors. J Biol Chem 2013, 288:13885-13896.
44. Gunasekera S, Daly N, Clark R, Craik DJ: Dissecting the oxidative folding of circular cystine knot miniproteins: Development of hybrid cyclotide scaffolds for protein engineering. Antioxid Redox Signal 2009, 11:971-980.
maar M, Siatskas C, Tagore P, Payne N, 61. Wang CK, Gruber CW, Ce Sun G, Wang S, Bernard CC, Craik DJ: Molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis. ACS Chem Biol 2014, 9:156-163.
45. Aboye TL, Clark RJ, Burman R, Roig MB, Craik DJ, Goransson U: Interlocking disulfides in circular proteins: toward efficient oxidative folding of cyclotides. Antioxid Redox Signal 2011, 14:77-86.
62. Chan Lai Y, Craik David J, Daly Norelle L: Cyclic thrombospondin-1 mimetics: grafting of a thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial cell migration. Biosci Rep 2015, 35:e00270.
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Current Opinion in Chemical Biology 2017, 38:8–16
16 Next generation therapeutics
63. Huang Y-H, Henriques ST, Wang CK, Thorstholm L, Daly NL, Kaas Q, Craik DJ: Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold. Sci Rep 2015, 5:12974. 64. Jagadish K, Gould A, Borra R, Majumder S, Mushtaq Z, Shekhtman A, Camarero JA: Recombinant expression and phenotypic screening of a bioactive cyclotide against a-synuclein-induced cytotoxicity in baker’s yeast. Angew Chem Int Ed 2015, 54:8390-8394. The first paper to report the recombinant expression of cyclotides inside live yeast cells. 65. Maaß F, Wustehube-Lausch J, Dickgiessr S, Valldorf B, Reinwarth M, Schmoldt HU, Daneschdar M, Avrutina O, Sahin U, Kolmar H: Cystine-knot peptides targeting cancer-relevant human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). J Pept Sci 2015, 21:651-660. 66. Sangphukieo A, Nawae W, Laomettachit T, Supasitthimethee U, Ruengjitchatchawalya M: Computational design of hypothetical new peptides based on a cyclotide scaffold as HIV gp120 inhibitor. PLoS One 2015, 10:e0139562.
75. Avrutina O, Schmoldt HU, Gabrijelcic-Geiger D, Wentzel A, Frauendorf H, Sommerhoff CP, Diederichsen U, Kolmar H: Head-to-tail cyclized cystine-knot peptides by a combined recombinant and chemical route of synthesis. ChemBioChem 2008, 9:33-37. 76. Austin J, Wang W, Puttamadappa S, Shekhtman A, Camarero JA: Biosynthesis and biological screening of a genetically encoded library based on the cyclotide MCoTI-I. ChemBioChem 2009, 10:2663-2670. 77. Getz JA, Rice JJ, Daugherty PS: Protease-resistant peptide ligands from a knottin scaffold library. ACS Chem Biol 2011, 6:837-844. 78. Glotzbach B, Reinwarth M, Weber N, Fabritz S, Tomaszowski M, Fittler H, Christmann A, Avrutina O, Kolmar H: Combinatorial optimization of cystine-knot peptides towards high-affinity inhibitors of human matriptase-1. PLoS One 2013, 8:e76956.
67. Aboye T, Meeks C, Majumder S, Shekhtman A, Rodgers K, Camarero J: Design of a MCoTI-based cyclotide with angiotensin (1-7)-like activity. Molecules 2016, 21:152.
79. Jagadish K, Borra R, Lacey V, Majumder S, Shekhtman A, Wang L, Camarero JA: Expression of fluorescent cyclotides using protein trans-splicing for easy monitoring of cyclotide–protein interactions. Angew Chem Int Ed 2013, 52:3126-3131. This paper showed that cyclotides containing the non-natural amino acid AziF can be labeled to monitor cyclotide-protein interactions.
68. Chan LY, Craik DJ, Daly NL: Dual-targeting anti-angiogenic cyclic peptides as potential drug leads for cancer therapy. Sci Rep 2016, 6:35347. An example of dual grafting of a cyclotide scaffold- in this case by grafting two anti-angiogenic epitopes targeting cancer.
80. Koehbach J, O’Brien M, Muttenthaler M, Miazzo M, Akcan M, Elliott AG, Daly NL, Harvey PJ, Arrowsmith S, Gunasekera S et al.: Oxytocic plant cyclotides as templates for peptide G proteincoupled receptor ligand design. Proc Natl Acad Sci U S A 2013, 110:21183-21188.
69. Conibear AC, Chaousis S, Durek T, Rosengren KJ, Craik DJ, Schroeder CI: Approaches to the stabilization of bioactive epitopes by grafting and peptide cyclization. Biopolymers: Pept Sci 2016, 106:89-100. 70. Swedberg JE, Mahatmanto T, Abdul Ghani H, de Veer SJ, Schroeder CI, Harris JM, Craik DJ: Substrate-guided design of selective FXIIa inhibitors based on the plant-derived Momordica cochinchinensis trypsin inhibitor-II (MCoTI-II) scaffold. J Med Chem 2016, 59:7287-7292. 71. Thell K, Hellinger R, Sahin E, Michenthaler P, Gold-Binder M, Haider T, Kuttke M, Liutkevi9 ciute_ Z, Go¨ransson U, Gru¨ndemann C et al.: Oral activity of a nature-derived cyclic peptide for the treatment of multiple sclerosis. Proc Natl Acad Sci U S A 2016, 113:3960-3965. A paper reporting oral activity for a cyclotide active in an animal model of multiple sclerosis.
81. Gray K, Elghadban S, Thongyoo P, Owen KA, Szabo R, Bugge TH, Tate EW, Leatherbarrow RJ, Ellis V: Potent and specific inhibition of the biological activity of the type-II transmembrane serine protease matriptase by the cyclic microprotein MCoTI-II. Thromb Haemost 2014, 112:402-411. 82. Senthilkumar B, Kumar P, Rajasekaran R: In-silico template selection of in-vitro evolved kalata B1 of Oldenlandia affinis for scaffolding peptide-based drug design. J Cell Biochem 2016, 117:66-73. 83. Huang YH, Colgrave ML, Clark RJ, Kotze AC, Craik DJ: Lysine-scanning mutagenesis reveals a previously unidentified amendable face of the cyclotide kalata B1 for the optimisation of nematocidal activity. J Biol Chem 2010, 285:10797-10805.
72. Kimura RH, Tran A-T, Camarero JA: Biosynthesis of the cyclotide kalata B1 by using protein splicing. Angew Chem Int Ed 2006, 118:987-990.
84. Craik DJ, Fairlie DP, Liras S, Price D: The future of peptide-based drugs. Chem Biol Drug Des 2013, 81:136-147. A recent review on the potential of peptides, including cyclotides, in drug development.
73. Werle M, Schmitz T, Haung H-L, Wentzel A, Kolmar H, Bernkop-Schnurch A: The potential of cystine-knot microproteins as novel pharmacophoric scaffolds in organ peptide drug delivery. J Drug Target 2006, 14:137-146.
85. Wang CK, Colgrave ML, Ireland DC, Kaas Q, Craik DJ: Despite a conserved cystine knot motif, different cyclotides have different membrane binding modes. Biophys J 2009, 97:1471-1481.
74. Aboye TL, Clark RJ, Craik DJ, Go¨ransson U: Ultra stable peptide scaffolds for protein engineering—synthesis and folding of the circular cystine knotted cyclotide cycloviolacin O2. ChemBioChem 2008, 9:103-113.
86. Burman R, Stromstedt AA, Malmsten M, Go¨ransson U: Cyclotide-membrane interactions: defining factors of membrane binding, depletion and disruption. Biochim Biophys Acta Biomembr 2011, 1808:2665-2673.
Current Opinion in Chemical Biology 2017, 38:8–16
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ScienceDirect Cyclotides as drug design scaffolds David J Craik and Junqiao Du Cyclotides are ultra-stable peptides derived from plants. They are around 30 amino acids in size and are characterized by their head-to-tail cyclic backbone and cystine knot. Their exceptional stability and tolerance to sequence substitutions has led to their use as frameworks in drug design. This article describes recent developments in this field, particularly developments over the last two years relating to the grafting of bioactive peptide sequences into the cyclic cystine knot framework of cyclotides to stabilize the sequences. Grafted cyclotides have now been developed that interact with protein or enzyme targets, both extracellular and intracellular, as well as with cell surface receptors and membranes. Address Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia Corresponding author: Craik, David J ([email protected])
its presence in a plant extract used as a uterotonic medicine in the Congo region of Africa in the 1960s. In that application women boiled the above-ground parts of Oldenlandia affinis plants to make a tea which was ingested during labour to accelerate childbirth [7]. This application implies two things: (i) that the peptide survives boiling; and (ii) it survives the digestive tract and is absorbed sufficiently to exert its uterotonic effect. The structure of kalata B1 was first determined in 1995 [8], revealing the CCK motif, which helped to explain the exceptional stability of kalata B1 and its associated favourable pharmaceutical properties. A wide range of other cyclotides have been discovered since in plants from the Violaceae, Rubiaceae, Cucurbitaceae, Solanaceae and Fabaceae plant families [2] and cyclotide sequences are documented and regularly updated in the publicly accessible database CyBase [9]. It is conservatively estimated that the cyclotide family might comprise more than 50 000 members [10].
Current Opinion in Chemical Biology 2017, 38:8–16 This review comes from a themed issue on Next generation therapeutics Edited by David Craik and Sonia Troeira Henriques
http://dx.doi.org/10.1016/j.cbpa.2017.01.018 1367-5931/ã 2017 Elsevier Ltd. All rights reserved.
Introduction Cyclotides [1,2] are disulfide-rich peptides from plants that have a head-to-tail cyclic backbone and cystine knot arrangement of three conserved disulfide bonds, which are linked CysI-CysIV, CysII-CysV and CysIII-CysVI. The combination of the cystine knot motif and cyclic backbone is referred to as a cyclic cystine knot (CCK) and is responsible for the exceptional stability of cyclotides. Their natural function is thought to be plant defence molecules, as they are expressed in a wide range of plant tissues and have activities against insects [3], nematodes [4], and molluscs [5], driven principally by their ability to form pores in membranes [6]. However, the exceptional stability of cyclotides has generated additional interest in their use in protein engineering and drug design applications. Figure 1 shows the sequence and structure of the prototypical cyclotide kalata B1, originally discovered based on Current Opinion in Chemical Biology 2017, 38:8–16
Several other properties, aside from their exceptional stability, have driven pharmaceutical interest in cyclotides. These include their tolerance to substitutions in their backbone loops, their potential for oral bioactivity [11], and most recently, the finding that some cyclotides are able to penetrate cells and thereby potentially interact with intracellular pharmaceutical targets [12,13]. There are currently 380 published papers primarily focused on cyclotides and much of this literature has been extensively reviewed over the last decade. Thus, here we do not attempt to discuss their discovery, natural functions, structural characterisation, or biosynthesis, which have been extensively reviewed elsewhere. (Table 1 provides a list of selected key reviews [14,15,16,17–23,24] from the last three years that cover these and other background topics). Rather, our focus here is solely on the use of cyclotides as frameworks in drug design [25–40]. These drug design applications have been underpinned by a number of fundamental studies on the synthesis and structural characterisation of cyclotides over the last decade, as schematically illustrated in Figure 1. Furthermore, studies on the cell penetration properties of cyclotides over the last few years have contributed to the possibility of using engineered cyclotides against intracellular pharmaceutical targets. The development of chemical methods for the synthesis of cyclotides has been centrally important to their applications as drug design scaffolds. Specifically, solid phase peptide synthesis and native chemical ligation [41,42,43–46] is routinely used to assemble the cyclic www.sciencedirect.com
Cyclotides as drug design scaffolds Craik and Du 9
Figure 1
Kalata B1
(a)
II
I
IV
III
VI
V
loop
2
lo
Cyclotide (CCK) scaffold
(b)
op
5
loop 6 V
loo VI p4
II III
lo
I
IV
1 op
loop 3
(c)
Synthesis
Structure
(31 papers)
(61 papers)
Information on targets and epitopes
Cell penetration (7 papers)
Intracellular targeting
Grafting (25 of 52 papers) Reviews
Framework optimisation
(16 papers)
(11 papers)
DRU NS G DES IGN APPLICATIO
Drug leads Current Opinion in Chemical Biology
Structure of the prototypic cyclotide kalata B1 and schematic overview of drug design applications. (a) Peptide sequence in one-letter amino acid code showing the disulfide connectivities. (b) Three dimensional structure (PDBID: 1NB1) showing the cystine knot arrangement of the three conserved disulfide bonds and the labelling of backbone loops between successive Cys residues. Cyclotides range in size form 28–37 amino acids, with the variation reflecting different loop lengths. It is estimated that the cyclotide family will comprise approximately 50 000 members [10]. (c) Schematic illustration of how recent fundamental studies on the synthesis, structures, and (for intracellular targets) cell penetration, have underpinned applications in drug design. The numbers of papers referred to in each box include papers published from 2006 onwards on the indicated topic.
backbone of cyclotides, which are well suited to this approach due to their multiple Cys residues. Recombinant methods have also been utilised to make cyclotides [47], as have chemoenzymatic cyclisation approaches [48,49]. These technologies typically are used to make engineered or grafted cyclotides in which one of the native loop sequences is replaced by a loop having a desired bioactivity. Structural studies have been important in the design and validation stages of these applications. Such structural studies of cyclotides have mainly involved NMR methods and there are currently 49 entries for cyclotides or related molecules in the PDB, 42 of which were determined via NMR and seven with X-ray crystallography. www.sciencedirect.com
Over the last decade there have been 52 papers in the literature focusing primarily on drug design applications of cyclotides, including 25 ‘grafting’ papers [11,12 ,13,50–70,71] where the aim is to introduce a desired activity into a cyclotide framework, 11 papers concerning the synthesis/optimisation of the CCK framework [72– 78,79,80–82] and 16 reviews on drug design applications of cyclotides [25–40]. This information is summarised in Figure 1, which provides a schematic illustration of how inputs from fundamental studies of cyclotide synthesis, structure and cell penetration, combined with inputs from the literature on the sequences of bioactive peptides have underpinned applications used to design grafted cyclotides to hit specific targets. Current Opinion in Chemical Biology 2017, 38:8–16
10 Next generation therapeutics
Table 1 Selected recent reviews on cyclotides Title
Topic General review Historical accounts Discovery Sequencing Structure Bioactivity Synthesis Biosynthesis Membrane binding Membrane binding Biotechnological applications Drug design applications
Ref.
Chemistry and biology of cyclotides: circular plant peptides outside the box Joseph Rudinger memorial lecture: discovery and applications of cyclotides Discovery, structure, function, and applications of cyclotides: circular proteins from plants Primary structural analysis of cyclotides Structural studies of cyclotides Circling the enemy: cyclic proteins in plant defence Chemical and biological production of cyclotides Cyclotide biosynthesis Importance of the cell membrane on the mechanism of action of cyclotides The increasing role of phosphatidylethanolamine as a lipid receptor in the action of host defence peptides Cyclotides in a biotechnological context: opportunities and challenges There have been sixteen reviews on this topic in since 2006, with most focussing in the use of the CCK as a scaffold, including three [38–40] post 2014 reviews
Figure 2 summarises the grafting concept and highlights that a variety of target receptor types and cellular locations of targets have been explored, ranging from extracellular enzymes to cell surface receptors, membranes, and even intracellular targets, including kinases [13] or protein:protein interactions [12]. In all of these applications just two subfamilies of cyclotide frameworks have been used. Cyclotides are actually divided into three subfamilies, the Mo¨bius, bracelet, and trypsin inhibitor subfamilies. Mo¨bius and bracelet cyclotides are most similar to each other, differing mainly by the presence or absence of a cis-Pro peptide bond in loop 5 of the backbone sequence whereas the trypsin inhibitor cyclotides have quite different sequences. Because bracelet cyclotides are more difficult to correctly fold, almost all grafting studies have focused on Mo¨bius or trypsin inhibitor scaffolds. The fact that bracelet cyclotides cannot practically be used as grafting frameworks is not a major limitation, given the availability of the two other complementary scaffolds, but the development of efficient folding pathways for bracelet cyclotides in future would be valuable to evaluate their potential applications. Recent studies have attempted to unravel the reasons for the difficult folding of bracelet cyclotides by examining chimeric bracelet/Mo¨bius cyclotides [44–46] and a range of other mutated cyclotides. Table 2 provides an analysis of the grafted cyclotides that have been reported in the literature to date, showing the epitope sizes and grafting locations (i.e., loops) used for the two utilised CCK framework subfamilies (Mo¨bius and trypsin inhibitor). The former subfamily framework tends to be used for extracellular targets and for cases when membrane binding might be advantageous, and the latter for intracellular targets or those involving enzyme inhibition. The table emphasises the vast range of applications of grafted cyclotides, from cancer to metabolic disease, pain and multiple sclerosis. It also emphasises the versatility of the CCK framework and its tolerance to loop Current Opinion in Chemical Biology 2017, 38:8–16
[18] [16] [24] [19] [20] [14] [22] [17] [15] [23] [21] [25–40]
substitutions of vastly varying content and size. For example, each and every one of the six backbone loops of the CCK framework has been used to incorporate bioactive epitopes, and the sizes of inserted epitopes have ranged from 1 to 21 amino acids (Table 2). We note that the grafts involving just single residue substitutions can equivalently be regarded as point mutations, and that such mutations have, in some cases, been used to optimize properties of the framework rather than introducing a target bioactivity per se. For example, single Lys substitutions in kalata B1 were used to increase membrane targeting for enhancing the nematocidal activities of cyclotides [83]. Nevertheless, in Table 2 we classify selected examples of these single-residue substitutions as examples of ‘grafting’ to highlight the generality and limits of the grafting approach. It is clear from Table 2 that most grafting studies have focused on single loop grafts, with typical epitope sizes of 6–8 amino acids. Loop 6 has been the most commonly grafted loop and loop 6 grafted cyclotides typically fold well for either Mobius or trypsin inhibitor frameworks. Loop 1 has so far only been used as a grafting site for trypsin inhibitor cyclotides, mainly owing to the fact that this is the reactive site loop of MCoTI peptides and thus naturally accommodates protease inhibitory activity, which can be tweaked for quite a wide range of different proteases. For example, loop 1 grafts have been used to make inhibitors of matriptase, foot-and-mouth-disease virus 3C protease, b-tryptase, Bcr-Abl kinase and FXIIa (Table 2). Other trends apparent from Table 2 are that, so far, loops 2 and 3 have been relatively underrepresented in grafting studies, and that it is possible to include unnatural chirality in grafts. For example, the development of agonists of the MCR4 receptor as potential treatments for obesity involved insertion of the epitope GHfRWG into loop 6 of kalata B1 (‘f’ denotes D-Phe). It is also worth noting that this example illustrates that
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Cyclotides as drug design scaffolds Craik and Du 11
agonists, as well as antagonists of receptors, can be developed using cyclotide grafting technology. Although most grafting studies have involved single loop substitutions, the simultaneous grafting of two or more of the six CCK loops is possible, and has been used in cases where the target epitope is particularly large, or in cases where dual targeting or dual functionality is desired [68 ]. An example of the former is the grafting of sequences from myelin oligodendrocyte glycoprotein (MOG) into kalata B1 for the development of drug leads for multiple sclerosis [61]. In that case, a 22-amino acid epitope spanned both loops 5 and 6 of kalata B1, with a Cys residue incorporated into the grafted epitope in a position that allowed the native disulfide bond to be retained in the CCK scaffold. An example of dual targeting was recently reported for an anti-angiogenic cyclotide, with Figure 2
(a) cell surface
(d) intracellular
(c) extracellular
(b) membrane
Grafting
Grafting Bioactive epitope(s)
kalata B1
MCoTI-II
Stable cylotide frameworks
the epitopes YwKV (‘w’ denotes D-Trp) and YHLNQPF grafted into loops 5 and 6, respectively, of MCoTI-II [68]. In our opinion, such dual targeting will be an area of increasing application in future, and represents a particular advantage of the cyclotide framework over some other peptide-based modalities. More complex grafting approaches involving modification of the cystine knot core or cyclic backbone are also possible in combination with grafting. For example, one study to develop a NS2B-NS3 Dengue protease inhibitor involved the use of a two-disulfide kalata B1 variant in which the CysII-CysV disulfide bond was removed via Cys residue substitutions and grafts were effectively inserted into loops 2 and 5, along with single point mutations in other loops. The authors of that study reported the presence of multiple isomers of the resultant two-disulfide product, and the exact structure of the final product was unclear, but at least the principles of simultaneously using several different engineering approaches is illustrated by this study [54]. Another example involved the use of a hybrid cystine knot peptide that combined two different inhibitory sequences and was cyclised via hydrazone formation. This engineered cyclotide had high inhibitory activity against trypsin (Ki = 0.1 nM) and tryptase (Ki = 1 nM) [55]. The size of cyclotides (around 30 amino acids, with a MW of 3000 Da) fills a gap between traditional small molecule drugs (MW <500 Da) and larger biologics (MW >5000 Da) such as antibodies [84] and to some extent this gives them some advantages over both: being bigger than small molecules they are more suited for blocking protein: protein interactions, and being smaller than antibodies they have better cell penetrating properties. Thus, cyclotides potentially can access targets that are inaccessible to either small molecules or antibodies. Of particular current interest is the use of cyclotides as carriers to deliver bioactive cargoes inside cells. The first published example exemplifying this principle involved the use of a grafted trypsin inhibitor cyclotide (MCoTI-I) to modulate the p53 HDM2 intracellular protein-protein interaction to reduce tumour growth in a mouse xenograft model [12]. Another recent example was the use of grafted MCoTI-II to targeting the intracellular SET protein implicated in leukaemia [13].
Current Opinion in Chemical Biology
Schematic illustration of the grafting process to highlight the diverse range of targets accessible to cyclotides. These targets range from: (a) cell surface receptors [51], to (b) cell membranes [15], to (c) extracellular targets (e.g., FMDV 3C protease [52]), to (d) intracellular targets [12,13]. Two types of cyclotide scaffolds are shown: kalata B1 as a representative of the Mo¨bius subfamily, and MCoTI-II as a representative of the trypsin inhibitor subfamily. The grafting process involves insertion of a bioactive epitope into one of these stable CCK frameworks. In the examples shown, loop 6 has been used to incorporate a bioactive helix. www.sciencedirect.com
Finally, since the natural function of cyclotides is as host defence agents, with a common mechanism of action involving membrane binding and disruption, there is an opportunity to use controlled membrane targeting of cyclotides for peptide epitope delivery. Recent studies have shown that the membrane binding of kalata B1 is mediated by interactions with phosphatidylethanolamine [15] and thus lipid selectivity might in principle be used to selectively target cyclotides to cells with particular lipid compositions on their surface. Supporting the proposal of Current Opinion in Chemical Biology 2017, 38:8–16
Summary of published grafting studies using CCK framework as a drug design scaffold. Biological activity Mo¨bius subfamily
Grafted sequence
5
VEGF-A antagonist
2 3 3 5 5 6 2&5
DK KNK RRKRRR RRKRRR NTRRKRRRG RRKRRR RRKRRRV RRKRRR SEESRRG&RRSR c
6 6 6 6 6 6 5&6
GHFRWG GHfRWGe HFRW HfRWe KRPPGFSPL QIPGLGPL KPLR& KAPRMVR i
Experimental autoimmune encephalomyelitis
5 5 6 5&6 4 2
RSPFSRV 6 others 6 others 4 others K K
HIV gp120 inhibitor
5&6
Sl&GSFLRFLTKG
Dengue NS2B-NS3 protease inhibitor Melanocortin 4 receptor (MC4R) agonist
Bradykinin B1 receptor antagonist Neuropilin-1/-2 antagonist
Immunomodulation (Experimental autoimmune encephalomyelitis, EAE, mouse model)
Trypsin inhibitor subfamily
Loop
Proof-of-concept studya
CXCR4 antagonist, HIV-1 cell entry blocker Hdm2/HdmX antagonist
FMDV 3C protease inhibitor
b-tryptase inhibitor b-tryptase and HLE inhibitor
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Angiogenic Trypsin and matriptase inhibitor CTLA-4 Synuclein-induced cytotoxicity
5&6 6 6 6 6 6 6 6 1 1 1 3, 5 & 6 3, 5 & 6 1 1 6 6 6 6 6 1, 3 & 6 1, 3 & 6 6
l
S &GSFLTGQGSF YRXCRGpRRBCYXKn 7 others GASRAPTSFAEYWNLLSA GASKAPTSFAEYWNLLSA GASKAPTSAAEYWNLLSA GASRAPTSAAEYWNLLSA GASRAPTSFAEYZNLLSAp Q AKQ F+3 others r LAG, GPNGF&AKKVHseZs LAG, GPNGF&SHseZDG s V 6 others SDGGl 7 others SIKVAV SVVYGLR R 15 others KYSHVP, K&PR 9 others SLATWAVG
Potency/dose
Application
–
Ref.
Confirm plasticity of CCK scaffold Anti-angiogenesis with the potential to reduce blood vessel growth in tumours.
[50]
1.39 0.35d 3.03 0.75 d 580 f
Dengue Fever
[54]
Obesity
[58]
49%g, 42%h 38%g, 28% h 100i f
Chronic pain & inflammatory pain Inhibit endothelial cell migration, angiogenesis, lymphangiogenesis Multiple sclerosis
[11]
12 b
28.116.6j
20 k
1067 m
[51]
[59]
[61]
Multiple sclerosis, T-cellmediated disorders
[71]
Anti-HIV
[66]
Anticancer and anti HIV-1
[57]
Inhibit tumour growth via modulation of intracellular protein-protein interaction
[12]
Foot-and-mouth disease
[52]
Allergic asthma and inflammation disorders Inflammatory disease
[55]
m
959 20f,o, 2f,o 2.3 0.1 , 9.7 0.9 q
2.6 0.4 q 41 25d 56 35d > 100 d 0.001d 4d >50d t, 0.021d 0.009
d t
q
t
, 0.0025
14.6u 13.9 u 0.00001d,v, 0.00029d,v 3.7
w
[53]
d t
Cardiovascular disease and/or wound healing Anticancer Immunotherapy of cancer and other diseases Explore the feasibility of phenotypic screening of bioactive cyclotides
[56] [60] [65] [64]
12 Next generation therapeutics
Current Opinion in Chemical Biology 2017, 38:8–16
Table 2
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Table 2 (Continued ) Biological activity
Loop
Grafted sequence
Potency/dose
EAIYAAPK&GEAIYAAPFAR 4 others 9 others 2 others GViTRIR x GNKRTRG GNKRTRG GNKRTRG GASKAPASXLRKLXKRLLRDAz GASKAPASXLRKLXKRLL z EIVYRXA
1.3 0.1
Angiotensin (1–7) receptor
1&6 1 6 1&6 6 1 2 6 6 6 6
Anti-angiogenic
5&6
1B,f
FXIIa and FXa inhibitors
1 1 1 1
YwKV&YHLNQPFB 6 others FR&Q SR&Q FRW&Q FRW&KQ
BCR-ABL kinase inhibitor
Cell migration inhibitor LyP1 marker for tumour lymphatics SET antagonist
& & & &
6 6 6 6
b
>128b,y >128/n.d. b,y >128 b,y 2.9 0.14b,z 5.0 0.15b,z
0.1C, 0.46C 2.16C, 96C 0.51C, >100C 0.49C, >100 C
Application
Ref.
Chronic myeloid leukaemia
[63]
Anti-angiogenesis Anti-tumour and anticancer
[62] [69]
Anticancer
[13]
Lung cancer or myocardial infarction Anticancer
[67]
Thrombosis, cardiovascular diseases
[68] [70]
a
Proof-of-concept study to demonstrate that loops are tolerant to modification. IC50, half maximal inhibitory concentration (mM). C9, C21 are substituted by E and R (bold), respectively; There are also mutations E7S, T8E, T20R and N29R in loops1, 4 and 6. The two isomers of grafted cyclotides have Kis of 1.39 0.35 mM (isomer B) and 3.03 0.75 mM (isomer C), respectively. d Ki, inhibition constant (mM). e Residue f refers to D-phenylalanine. f EC50, half maximal effective concentration (nM). g Inhibition of writhing after intraperitoneal injection (i.p. 1 mg/kg). h Inhibition of writhing under oral administration (p.o. 10 mg/kg). i There are mutations of V10 M in loop2 and N15R, P17F in loop3, the EC50 was tested on HUVEC cells. j Cumulative clinical score using experimental autoimmune encephalomyelitis mouse model was significantly lower than the cumulative score of control group. k Oral administration (mg/kg). l Truncated residue/s. m The binding affinity was determined by measuring the non-covalent interaction energy, which is a combination of electrostatic and VDW interaction energies (kJ/mol). n Single letter codes B, X, and p represent the amino acids 2-naphthylalanine, citruline, and D-proline, respectively. o A potent CXCR4 antagonist with EC50 20 nM and an efficient HIV-1 cell-entry blocker with EC50 2 nM. p Z = 6-chlorotryptophan. q Binding affinity of grafted cyclotides to recombinant Hdm2 and HdmX was measured by fluorescence polarisation anisotropy. Cyclotide MCo-PMI-K37R displayed strong affinity for Hdm2 (KD = 2.3 0.1 nM) and HdmX (KD = 9.7 0.9 nM); MCo-PMI-6ClW had affinity of Hdm2 (KD = 2.6 0.4 nM). r Another three cyclotides have mutations K10R, K10V, K10A. s Hse, homoserine; Z, N-terminal glyoxylyl residue. t MCoTI-II[K10V] displayed inhibitory activity of Ki >50 mM (tryptase) and Ki 0.021 mM (HLE); MCoTI-II[SDGG truncated] had inhibitory activity of Ki 0.009 mM (tryptase), and Ki 0.0026 mM (trypsin). u The lowest concentration with significant difference compared to control (mM). v MCoTI-II[V3R] displayed inhibition of Ki 0.00001 mM (trypsin) and Ki 0.00029 mM (matriptase). w Binding constant was estimated using ELISA(mM). x Letter i = D-Isoleucine. y Cytotoxicity of grafted cyclotide in loop 1and loop 6 against normal and cancer cell lines were same as IC50>128 mM (MDA-MB-435S, MDA-MB-231, MM96L, HFF1), the IC50 of grafted cyclotide in loop 2 was >128 mM in MDA-MB-435S and not determined in other three cell lines. z Letter X represents amino-isobutyric acid. The IC50 was tested against K562 cells. A Residue X represents L-2,3-diaminopropionic acid. B Residue w represents D-tryptophan, the EC50 was tested on HUVEC cells. C Inhibition constants of each grafted cyclotide for FXIIa and FXa are Ki 0.11 mM and 0.46 mM, 2.16 mM and 96 mM, 0.51 mM and >100 mM, 0.49 mM and >100 mM, respectively. b c
Cyclotides as drug design scaffolds Craik and Du 13
Current Opinion in Chemical Biology 2017, 38:8–16
14 Next generation therapeutics
selectivity in membrane targeting, another study showed that cyclotides from different subfamilies (Mo¨bius and bracelet) differed in their orientation when binding to membranes [85]. It has also been reported that cycloviolacin O2 (a bracelet cyclotide) has stronger cytotoxicity against various cancer cell lines than kalata B1 or kalata B2 (Mo¨bius subfamily members) and was more potent towards anionic DOPC/DOPA liposomes than zwitterionic DOPC, suggesting a degree of cell specificity related to the number of positive charged residues in the cyclotide framework [86].
Conclusions Cyclotides are now a widely-studied family of plant proteins and are gaining acceptance as potential drug design scaffolds. No cyclotides have yet reached human clinical trials but, in our opinion, there is a good possibility that this may occur in the not too distant future. The biggest challenge in the field at the moment is increasing the oral bioavailability of peptide-based leads. Although there are several reported cases where cyclotides have oral activity, there is limited literature on the actual oral bioavailability and so more quantitative studies reporting such biopharmaceutical parameters are expected over coming years. This information will make a valuable addition to the wealth of information currently available on the stability and pharmaceutical ‘graftability’ of cyclotides.
Acknowledgements Work on cyclotides in our laboratory is supported by the Australian Research Council (DP150100443) and the NHMRC, Australia (APP1084604). DJC is an ARC Australian Laureate Fellow (FL150100146).
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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16 Next generation therapeutics
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