Cyclotides as Tools in Chemical Biology - Accounts of Chemical

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Cyclotides as Tools in Chemical Biology Published as part of the Accounts of Chemical Research special issue “Chemical Biology of Peptides”. Simon J. de Veer, Joachim Weidmann, and David J. Craik* Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia CONSPECTUS: Among the various molecules that plants produce for defense against pests and pathogens, cyclotides stand out as exceptionally stable and structurally unique. These ribosomally synthesized peptides are around 30 amino acids in size, and are stabilized by a head-to-tail cyclic peptide backbone and three disulfide bonds that form a cystine knot. They occur in certain plants of the Rubiaceae, Violaceae, Cucurbitaceae, Fabaceae, and Solanaceae families, with an individual plant producing up to hundreds of different cyclotides. Aside from being exploitable as crop protection agents based on their natural pesticidal activities, cyclotides are amenable to repurposing by chemists for use as drug leads or as tools in chemical biology. Their macrocyclic peptide backbone and knotted arrangement of three disulfide bonds engenders cyclotides with resistance to proteolytic degradation, high temperatures, and chemical chaotropes. Furthermore, their small size makes them accessible to synthesis using solid-phase peptide chemistry and so nonnatural cyclotides can be designed and synthesized for a variety of applications. Our focus here is on cyclotides as tools in chemical biology, and there are four main areas of application that have appeared in the literature so far: (i) cyclotides as probes of membrane binding; (ii) cyclotides as probes of biosynthetic pathways for peptide cyclization; (iii) cyclotides as probes of protease specificity and function; and (iv) cyclotides as probes of receptor binding and specificity, with the potential for them to be developed as drug leads. The main methods used in these studies include solid-phase peptide chemistry for synthesis and NMR spectroscopy for structural characterization, as well as a wide range of biochemical and biophysical techniques for probing intermolecular interactions. In addition, cyclotides have been examined in diverse biological assays, ranging from enzyme inhibition to cell penetration, intracellular targeting and cytotoxicity. The main finding to have emerged from studies over the past decade is that cyclotides are exceptionally stable under a variety of conditions (in assay buffers, biological fluids, membranes, and recombinant expression systems). Furthermore, they are structurally very well-defined and amenable to sequence substitutions that can introduce new desired biological activities, generally without compromising their exceptional stability. Both features contribute to their use as peptide-based frameworks in drug design. Finally, they occupy a size niche between traditional small-molecule drugs (5000 Da) and thus can probe receptors, membranes, and protein−protein interactions in different ways to what is possible with either small molecules or biologics. Overall, cyclotides are an exciting class of peptides that have great potential as ultrastable chemical biology probes in a variety of applications. They have the advantage of specificity (typical of proteins) combined with the synthetic accessibility of small molecules.

1. INTRODUCTION

host defense activity by binding to and disrupting membranes in pest species.8 Cyclotides range from 28−37 amino acids in size, and incorporate a diverse range of sequences in their six backbone loops between the conserved Cys residues. Indeed, we estimate there to be at least 50 000 different sequences of naturally occurring cyclotides,9 although this number could be even higher. This diversity presumably evolved to target different cyclotides to different pests. Incidentally, cyclotide sequence diversity provided part of the motivation for our suggestion of cyclotides as drug design scaffolds, i.e. the natural tolerance to sequence substitutions suggested that it would be possible to synthesize modified cyclotides with sequence changes in one or

1

Cyclotides are plant-derived host defense peptides characterized by their head-to-tail cyclic backbone and cystine knot core (Figure 1). Their exceptional stability and resistance to proteolytic degradation have contributed to their use as drug leads2 and scaffolds for peptide-based drug design.3−6 These properties have also led to great interest in their potential application for crop protection.7 Cyclotides have been reported in ∼50 different plant species from five major plant families. They appear to be ubiquitous in plants of the Violaceae family, where every species examined so far contains cyclotides, but are sparsely distributed in other parts of the plant kingdom. Cyclotide-bearing plants contain up to hundreds of different cyclotides that are distributed in all tissues, including leaves, flowers, stems and roots.7 It is believed that they exert their © 2017 American Chemical Society

Received: March 31, 2017 Published: June 23, 2017 1557

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Figure 1. Cyclotide structural overview. The six conserved Cys residues (I−VI) that comprise the cystine knot and the six backbone loops (1−6) are indicated. Representative sequences from each of the three subfamilies, Möbius (kalata B1), bracelet (cycloviolacin O2, cyO2), and trypsin inhibitor (MCoTI-II), are shown. The main difference between Möbius and bracelet cyclotides is the cis-Pro peptide bond in loop 5 (highlighted in the inset). Also highlighted is loop 1, the active site loop of trypsin inhibitor cyclotides, and loop 6, where an AEP mediates cyclization of the peptide backbone.

peptide thioesters that undergo head-to-tail cyclization via intramolecular native chemical ligation (Figure 2A).24,26,25 Fmoc-based SPPS is a popular strategy to synthesize peptides as it involves milder conditions but, until recently, was less frequently used for cyclic peptides. The development of Fmoccompatible strategies for producing thioester peptides, including mildly activated N-acylurea thioester precursors27 and activating peptides assembled on sulfonamide resin,28 has made cyclotide synthesis more accessible, with cyclization achieved by intramolecular native chemical ligation similar to traditional Boc thiosters (Figure 2B). Another Fmoc-based strategy relies on the cyclization of protected peptide fragments29 (Figure 2C). Here, protected peptides are released from the resin by mild cleavage and cyclized at low concentrations in solution using HATU. Side chain protecting groups are subsequently removed, and purified cyclic peptides are folded by oxidation. In many cases, the three disulfide bonds that comprise the cystine knot can be correctly connected using air oxidation. If folding is problematic, other strategies can be tested including selective disulfide formation (e.g., Acm-protected thiols) or addition of organic solvents (e.g., 2-propanol). Depending on the cyclotide sequence, either Boc- or Fmocbased synthesis methods can give good results. The availability of two separate chemical strategies is also an advantage for troubleshooting, and in cases where a given cyclotide fails to assemble using one approach, the other can be trialed. Most Möbius and trypsin inhibitor cyclotides assemble reasonably well using Fmoc synthesis, and this strategy has now become our standard procedure as it involves less hazardous chemicals. Overall, chemical synthesis has facilitated a wide range of applications of cyclotides and has been pivotal for the elucidation of structure−activity relationships. Additionally, it has enabled facile sequence modification for cyclotide-based drug design and the production of synthetic substrates to study cyclotide biosynthesis. We now describe examples of these and other applications of cyclotides to probe biological processes and pathways.

more backbone loops. Aside from the conserved Cys residues, a highly conserved Asn/Asp is important for processing of cyclotide precursor proteins by asparaginyl endopeptidases (AEPs), which are involved in the biosynthetic cyclization of cyclotides.10−12 This Asn/Asp is the C-terminal residue of the cyclotide domain in the precursor protein, and becomes incorporated into loop 6 of the mature cyclotide. Three subfamilies of cyclotides have been identified to date: Mö bius, bracelet, and trypsin inhibitors. The first two subfamilies differ in the presence (Möbius) or absence (bracelet) of a cis-Pro peptide bond in the cyclic peptide backbone. About one-third of natural cyclotides fall into the Möbius subfamily and two-thirds into the bracelet subfamily. The trypsin inhibitor cyclotides comprise only a few dozen members, all derived from the seeds of tropical vines in the Momordica genus.13 In contrast with the Möbius and bracelet cyclotides, trypsin inhibitor cyclotides do not have insecticidal activity, and we assume their natural function is to inhibit digestive enzymes in the guts of seed-dispersing animals. Sequences of representative members of each subfamily are shown in Figure 1. The discovery,14,15 structural characterization,16 bioactivities,17 and applications of cyclotides as drug design scaffolds18−21 have been extensively reviewed recently, and readers are referred to these articles and a book22 on cyclotides for a detailed background, and to the online database, CyBase,23 for a listing of cyclotide sequences. In this Account, our focus is on cyclotides as tools in chemical biology, a topic that has not yet been reviewed. We first discuss methods available for the chemical synthesis of cyclotides as these underpin applications in chemical biology.

2. SYNTHESIS Solid-phase peptide synthesis (SPPS) allows the assembly of cyclotide sequences on the milligram scale and thus complements extraction methods as a source of native cyclotides and provides access to non-native cyclotides. Figure 2 gives an overview of the two main strategies for chemical cyclotide synthesis; i.e., Boc- or Fmoc-based chemistries, and the advantages and limitations of each method have been covered in a recent review.24 In our laboratory, Boc-based SPPS of cyclotides utilizes the in situ neutralization protocol to produce

3. APPLICATIONS IN CHEMICAL BIOLOGY Figure 3 outlines how cyclotides have contributed as tools in chemical biology. In summary, the four major fields of 1558

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Figure 2. Solid-phase peptide synthesis of cyclotides. (A) The Boc approach employs thioester peptides25,26 that are first cleaved (1), then cyclized through autoligation (2), and subsequently oxidized (3) to the folded cyclotide. (B) The Fmoc N-acylurea strategy2,27 is an example of an Fmoccompatible approach for synthesizing thioester peptides and is commonly performed on 3-4-diaminobenzoic acid Rink amide resin. After peptide assembly, the C-terminus is activated to a N-peptidyl-urea (4) followed by cleavage and purification (5). The N-peptidyl-urea is subsequently converted to a thioester by thiolysis and cyclized (6). (C) The Fmoc-based side chain-protected approach29 involves peptide assembly on chlorotrityl resin followed by mild cleavage (7), cyclization of the side chain-protected peptide in solution (8), and deprotection (9). Irrespective of the method for chain assembly, disulfide oxidation is typically performed using 1 mM reduced peptide in aqueous ammonium bicarbonate (pH ∼ 8). Addition of organic solvents (e.g., 2-propanol) or reduced and oxidized glutathione is beneficial in some cases.

structure of kalata B1 to form a “bioactive face”, which is distinct from a “hydrophobic face” made up of other residues (Figure 4B). A direct link between bioactivity and membrane interaction was later established by studying the binding of kalata B1 mutants to different lipids using surface plasmon resonance.32 That study revealed that kalata B1 has a very specific mechanism for binding to membranes, which is driven by interaction with phospholipids containing phosphatidylethanolamine (PE) head groups (Figure 4C). Mutants that displayed higher or lower bioactivity than kalata B1 had a proportional increase or decrease in PE binding affinity.32 Membrane targeting via PE interaction appears to be a common feature among other cyclotides from the Möbius and bracelet subfamilies. Naturally occurring cyclotides from both subfamilies bind to membranes that contain PE phospholipids and induce membrane leakage in PE-containing vesicles, but have much lower activity when PE is absent.33 Collectively, these studies demonstrate how native and chemically synthesized cyclotides with specific amino acid

application are (i) understanding aspects of membrane biology; (ii) exploiting enzymes for peptide cyclization; (iii) probing enzyme specificity to generate cyclotides that target enzymes of pharmaceutical interest; and (iv) using cyclotides to probe receptor biology, with the ultimate aim of developing cyclotidebased drugs. 3.1. Using Cyclotides to Explore Peptide-Membrane Interactions and Membrane Biology

Since many of the biological activities of cyclotides involve membrane interactions, a range of native and mutated cyclotides have been used as chemical probes to study the molecular basis by which this interaction occurs. Initial studies focused on kalata B1 and compared its hemolytic or insecticidal activity to that of various mutants, including Ala or Lys point mutants, and acyclic permutants.30,31 These studies identified six residues that are particularly important for biological activity. Despite occurring in three different loops (Figure 4A), all six residues cluster together in the three-dimensional 1559

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Figure 3. Uses of cyclotides as tools in chemical biology. Four main areas include their use to probe membrane biology, biosynthetic cyclization pathways, proteolytic enzyme function, and receptor binding selectivity. The central panel shows a stylized drawing of the prototypic cyclotide kalata B1. This cyclotide binds to membranes (upper left) via a specific interaction with phosphatidylethanolamine phospholipids (blue). The biosynthetic pathway from gene to precursor protein to mature cyclotide is illustrated on the upper right and involves two enzymatic processing steps, with the second mediated by an asparaginyl endopeptidase (AEP), Probing enzyme function is represented on the lower left using the structure of the prototypic trypsin inhibitor cyclotide MCoTI-II bound to trypsin (PDB ID: 4gux). Probing receptor selectivity is illustrated on the lower right using a grafted cyclotide that selectively targets the melanocortin-4 receptor (MC4R).

Figure 4. Interaction between kalata B1 and membranes revealed using chemically synthesized peptides. (A) Kalata B1 schematic showing residues in the bioactive face (purple), hydrophobic face (green) and cystine knot (yellow). Hexagons indicate residues that can be substituted to produce kalata B1 mutants with increased bioactivity (named the amendable face). (B) Kalata B1 structure (surface representation, PDB ID: 1nb1) illustrating the bioactive and hydrophobic faces. The interaction between the bioactive face (centered around Glu7) and the positively charged headgroup of a phosphatidylethanolamine (PE) phospholipid is shown on the right. (C) Location of PE phospholipids (blue) within a lipid bilayer.

phosphates, consistent with observations that Lys to Ala mutations in MCoTI-II lead to lower internalization efficiency.36 These studies on the cell penetration of cyclotides open the possibility of using them as carriers to deliver cargoes to intracellular targets for pharmaceutical applications (subsection 3.4). More broadly, the ability of cyclotides to bind to specific lipids and cross membranes provides exciting opportunities for cell biology applications. For example, regions of a cyclotide that are important for membrane interactions could be engineered to shift its binding specificity to different lipids. Additionally, features from cyclotides could be transferred to other peptides to equip them with membrane-binding activity.

substitutions can be applied to probe mechanisms that underpin biological functions. In other applications of cyclotides as chemical biology tools, specialized nonpeptidic chemical moieties have been introduced into cyclotides to probe membrane interactions (Figure 5). These include trifluoromethylbicyclopentylglycine moieties to determine the orientation of cyclotides in membranes,34 ester moieties to examine how the charge of the conserved Glu in loop 1 affects bioactivity,35 Arg-modifying reagents to do likewise,35 and fluorescent dyes to probe vesicles, cells, and intracellular targeting.36 The ability to couple cyclotides with chemical dyes has also facilitated the discovery that some cyclotides can penetrate cells.36 For kalata B1, internalization is initiated by interactions between its bioactive face and PE phospholipids, followed by insertion of its hydrophobic face into the membrane.37 Interestingly, the trypsin inhibitor cyclotides MCoTI-I and MCoTI-II, which lack the ability to bind to PE, can also internalize into cells.36,38 Here, cellular uptake appears to involve interaction with negatively charged phosphatidylinositol

3.2. Synthetic Cyclotide Precursors for Probing Biosynthesis Cyclization Mechanisms

Cyclotides are ribosomally synthesized as precursor proteins that contain one or more cyclotide domains. These precursors undergo enzymatic processing to generate mature cyclotides, with AEPs being one class of enzymes that has a critical role in cyclotide maturation.10 Accordingly, synthetic cyclotide pre1560

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ability to perform site-specific ligation or cyclization reactions for diverse peptides and proteins. The processing and cyclization of kalata B1 was recently investigated using such chemically synthesized peptides and AEP1b from Oldenlandia affinis (OaAEP1b), which was recombinantly expressed in Escherichia coli.12 These assays revealed that OaAEP1b correctly processed and cyclized a kalata B1 precursor that contained its C-terminal propeptide (TRN↓ GLPSLAA) and a precursor that lacked disulfide bonds (each cysteine mutated to serine). However, OaAEP1b could not cyclize a precursor that also contained an N-terminal propeptide (LQLK↓GLP), indicating that cyclization is influenced by the sequence of the N-terminal (incoming) segment and that the N-terminal propeptide is released by a different enzyme.12 Additionally, cyclization assays were performed in the presence or absence of 18O-labeled water to test whether C-terminal cleavage is directly coupled to cyclization, or whether there is an intervening hydrolysis step. No isotopic shift was observed when the reaction products were analyzed by mass spectrometry, which is consistent with hydrolysis not occurring between cleavage and cyclization.12 However, these data do not definitely rule out hydrolysis during the cyclization reaction and the mechanism for AEP-mediated cyclization remains a topic of investigation and debate. Cyclotide biosynthesis has also been investigated using butelase 1, an AEP purified from Clitoria ternatea (butterfly pea) seedpods.11 The native enzyme was screened against a range of synthetic peptides, including kalata B1 precursors, to assess sequence requirements for efficient AEP-mediated cyclization. It cyclized kalata B1 precursors when the Cterminal propeptide was modified to match precursors found in butterfly pea (TRN↓HVIA), which could be truncated to (TRN↓HV) without affecting cyclization.11 However, further truncations or substitution of the Asn residue in the cyclotide domain prevented efficient cyclization. Butelase 1 was also screened against a library of synthetic peptides to profile its specificity in a peptide ligation assay. This assay demonstrated that butelase 1 has relatively broad specificity at the P1′ position (corresponding to the N-terminal residue of the incoming peptide segment), but more restricted specificity at P2′ where Leu, Ile, and Val were preferred.11 Several of these findings correlated with previous studies of kalata B1 cyclization in transgenic plants, including the importance of a branched hydrophobic residue at P2′ (Leu or Ile) and that the minimum length of the C-terminal propeptide is two residues.10 Indeed, both chemical and biological approaches are complementary, with experiments using synthetic peptides and purified enzymes providing faster assays and access to a higher degree of chemical diversity. With expanding knowledge on the activity and specificity of different AEPs, they are increasingly being explored for a wide range of protein engineering applications. For example, an OaAEP1b mutant that acts as a highly efficient ligase was recently reported and shown to conjugate several diverse peptides.39

Figure 5. Cyclotide-based chemical tools for probing membrane binding and bioactivity. Different types of modifications are illustrated on a kalata B1 schematic (basic residues, blue; acidic residues, red; remaining residues in the bioactive face, purple). Chemical probes can be produced by substituting a label in place of an amino acid; for example, substituting Val with trifluoromethyl bicyclopentylglycine to produce a probe for analyzing interactions with various biomolecules. Additionally, an amino acid can be substituted with Lys to enable conjugation of different tags, including biotin or Alexa Fluor 488, in order to track cyclotide-binding interactions or cellular uptake. Chemical probes can also be generated from cyclotides isolated from plants where site-selective chemistry is used to modify certain residues; for example, chemical modification of Glu or Arg in cycloviolacin O2.

cursors and purified AEPs have been used to explore the mechanisms for enzymatic processing and cyclization (Figure 6). At present, this topic also attracts major interest from outside the cyclotide field, and AEPs are becoming increasingly investigated for protein engineering applications due to their

Figure 6. Exploring cyclotide biosynthesis using chemical tools. The arrangement of the Oak1 precursor protein harboring the prototypic cyclotide kalata B1 (green) is shown, along with other key domains: endoplasmic reticulum signal sequence (ER, blue), N-terminal propeptide (NTPP, yellow) and C-terminal propeptide (CTPP, orange). The mechanism for processing and cyclization of kalata B1 has been investigated using a range of synthetic peptides based on Oak1. Here, synthetic peptides containing different substitutions can be tested against AEP enzymes that have been recombinantly expressed or purified from plants. Products from these reactions can be analyzed to determine how specific substitutions affect enzymatic processing or cyclization.

3.3. Engineered Cyclotides for Investigating the Biological Functions of Proteases

Cyclotides are very useful chemical scaffolds for targeting specific proteases. Such scaffolds have a range of applications, including blocking the activity of individual proteases to determine their contribution to biological processes, or as labeled probes for detecting a given protease in extracts, cultured cells or tissues. Cyclotides are particularly suited to 1561

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Accounts of Chemical Research 3.4. Receptor Specificity and Grafting

targeting proteases because they are much more resistant to proteolytic degradation than linear peptides, and show high affinity and selectivity due to their well-defined topology and multiple binding loops. Most of the work in this area has focused on trypsin inhibitor cyclotides. As trypsin belongs to a large family of structurally similar proteases, these cyclotides are a useful starting point for designing new inhibitors for many of these enzymes. Figure 7 shows the repertoire of proteases that have been targeted by engineered cyclotides. To date, three approaches

Membranes and enzymes represent just two classes of targets for cyclotides. Generally, applications in these areas harness the cyclotide’s existing biological activity and the goal is to redirect it to a new target or process. Another major application of cyclotides has been as scaffolds for the incorporation of bioactive peptide sequences directed toward specific receptor targets. This approach stabilizes the peptide epitope and, at the same time, equips the cyclotide with a new biological activity. Figure 8 illustrates this concept: although a linear bioactive

Figure 7. Cyclotides as scaffolds for targeting different proteases. Examples are shown of sequence substitutions that have been performed in loops 1, 2, or 6 for MCoTI-II or kalata B1. The amino acid sequence of the native cyclotide is circled in blue and modified sequences are shown underneath the parent sequence (substituted residues, black; unchanged residues, gray). XX indicates a homoserine-glyoxylyl motif that was used for backbone cyclization.

Figure 8. Overview of cyclotide “grafting”. The grafted epitopes typically comprise 6−8 amino acids (depicted as jigsaw pieces). Incorporating the epitope into a cyclotide framework prevents its degradation.

have been used to develop protease-targeting cyclotides. The first uses synthetic chemistry to change the sequence of an existing cyclotide. Here, sequence substitutions can be identified by screening the protease against synthetic peptide libraries, molecular modeling, or using known specificity data from related inhibitors. Inhibitors of factor XIIa and matriptase have been developed using this method,5,40 with most attention given to loop 1 as this is the primary binding loop of trypsin inhibitor cyclotides. The second approach focuses on modifying the structure of an existing cyclotide scaffold prior to sequence optimization. This strategy was used to produce inhibitors for β-tryptase by decreasing the number of residues in loop 6 or designing a chimeric inhibitor where part of the cyclotide is replaced with a segment from a structurally similar protease inhibitor.41,42 The third strategy is based on displaying libraries of acyclic variants on the surface of bacterial or yeast cells. Here, specific residues in the scaffold are randomized using degenerate codons and new inhibitors are identified following successive rounds of selection and enrichment. Due to the high degree of sequence diversity that can be achieved, this approach has been applied to cyclotides beyond the trypsin inhibitor family; for example, engineered cyclotides targeting thrombin or matriptase have been developed based on kalata B1 or MCoTI-II, respectively.43,44 As the number of engineered cyclotides that target proteases continues to grow, their use as probes for exploring the biological functions of proteases is expanding. One recent example illustrating this concept used an engineered MCoTI-II variant to block activation of pro-uPA on the surface of prostate cancer cells by inhibiting matriptase.44 Although few studies have progressed beyond biochemical assays, this remains an area of active investigation. Indeed, compared to other serine protease inhibitors, cyclotides occupy a relatively rare chemical space as they contain multiple binding loops that can be customized to target a specific protease.

peptide might be very potent, it typically will be susceptible to proteolysis and thus may not reach its target if administered systemically. By contrast, incorporating the bioactive epitope into a cyclotide scaffold can protect it from degradation. There are now more than two dozen published examples in which scaffolds based on either kalata B1 or MCoTI-II have been used as grafting frameworks, with applications including tumor suppression,3,45 leukemia,46 cardiovascular disease,47 HIV,48 multiple sclerosis,4 inflammatory pain,6 and obesity.49 The topic of cyclotide grafting has been extensively reviewed18−21,24 so we will not expand here, except to focus on one aspect relevant to chemical biology; over the course of grafting studies it has become clear that grafted cyclotide frameworks can be used to selectively distinguish between closely related receptors. The example illustrated in Figure 3 concerns melanocortin receptors (MCRs), where we designed a selective MC4R agonist as a potential treatment for obesity. The engineered cyclotide incorporating the bioactive tetrapeptide HFRW had high affinity (KD 29 nM) for MC4R with at least 300-fold selectivity over MC1R, MC3R, and MC5R.49 We foreshadow that there will be increasing use of cyclotides in the future as tools for probing receptor subtype selectivity. Indeed, a recent study reported that kalata B7 can be used to design peptides that target two further GPCRs, the oxytocin receptor and the vasopressin V1a receptor, with a peptide derived from loop 3 showing selectivity for the oxytocin receptor.50 Another area of increasing application is likely to be in grafting studies directed at intracellular targets. So far, intracellular targeting has mostly been studied in the context of cancer therapy targeting either protein−protein interactions or intracellular enzymes. For example, the p53−HDM2 interaction was blocked using a grafted cyclotide,3 and grafted 1562

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Accounts of Chemical Research cyclotides targeting the BCR-ABL kinase, implicated in chronic myeloid leukemia, were active in vitro.46

Joachim Weidmann undertook Ph.D. studies at The German Cancer Research Centre in Heidelberg, and The Scripps Research Institute in La Jolla, California, before joining UQ as a Research Assistant. His research focuses on application of peptide chemistry with the objective to answer biologically significant questions.

4. CONCLUSIONS AND OUTLOOK From the studies described herein, it is clear that cyclotides are exceptionally stable and amenable to chemical manipulation to vary their sequences, attach chemical or imaging probes, and use them for probing enzyme specificity or membrane biology. Undoubtedly, they can be regarded as useful chemical biology tools. Most previous efforts in the field of chemical biology have used small molecules as chemical probes for interrogating biological phenomena. Cyclotides are several-fold bigger and incorporate a different mix of chemical diversity than small molecules, thus giving them complementary advantages. Their use so far has been directed mainly at pharmaceutical applications. Although at ∼30 amino acids cyclotides are formally classified as peptides, we regard them as mini-proteins that display some of the characteristics of larger proteins, in that they have well-defined elements of secondary structure and stable three-dimensional structures. However, with molecular weights of ∼3000 Da, they fill a gap in the pharmaceutical industry between traditional small molecule drugs (5000 Da). It is thus their size and exceptional stability that allow cyclotides to capture the best of the two worlds of small molecules and protein-based-biologics. The ability to penetrate into cells is taken for granted in many small molecule drugs, but is far from the norm for peptides or proteins. In this regard, cyclotides are special and have been unequivocally demonstrated to enter the cytoplasmic space to target intracellular proteins. The challenge now in chemical biology is to quantif y the extent of cellular penetration, i.e., to measure intracellular concentrations, compartmentalization, and breakdown. Other challenges include accelerating the speed and reducing the cost of cyclotide synthesis. In this area, considerable progress has been made with mutant cyclotide libraries, using either synthetic chemistry28 or expression directly in cells,51 and these approaches will assist the design of additional cyclotide variants with new biological activities. We see these topics not as challenges, but as opportunities that we hope will encourage a wider range of researchers to investigate these fascinating molecules, so that their potential may be explored across an even broader range of chemical biology applications.



David J. Craik is a Professor of Chemistry at IMB, UQ. He obtained his Ph.D. in organic chemistry from La Trobe University in Australia and undertook postdoctoral studies at Florida State and Syracuse Universities (USA), before taking up a lectureship at the Victorian College of Pharmacy in 1983. He was appointed Professor of Medicinal Chemistry in 1988. He moved to UQ in 1995 to set up a biomolecular NMR laboratory and is currently an Australian Research Council (ARC) Laureate Fellow. His research focuses on applications of cyclic peptides, toxins, and NMR in drug design. He is a Fellow of the Australian Academy of Science and has received numerous awards for his research, including the Hirschmann Award from the American Chemical Society.



ACKNOWLEDGMENTS Work in our laboratory on cyclotides is supported by grants from the NHMRC (APP1107403) and the ARC (DP150100443). D.J.C. is an ARC Australian Laureate Fellow (FL150100146), and S.J.d.V. is a NHMRC Early Career Fellow (APP1120066).



REFERENCES

(1) Craik, D. J.; Daly, N. L.; Bond, T.; Waine, C. Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J. Mol. Biol. 1999, 294, 1327−1336. (2) Thell, K.; Hellinger, R.; Sahin, E.; Michenthaler, P.; Gold-Binder, M.; Haider, T.; Kuttke, M.; Liutkevičiu̅tė, Z.; Göransson, U.; Gründemann, C.; Schabbauer, G.; Gruber, C. W. 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. (3) Ji, Y.; Majumder, S.; Millard, M.; Borra, R.; Bi, T.; Elnagar, A. Y.; Neamati, N.; Shekhtman, A.; Camarero, J. A. In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide. J. Am. Chem. Soc. 2013, 135, 11623−11633. (4) Wang, C. K.; Gruber, C. W.; Č emažar, M.; Siatskas, C.; Tagore, P.; Payne, N.; Sun, G.; Wang, S.; Bernard, C. C.; Craik, D. J. Molecular grafting onto a stable framework yields novel cyclic peptides for the treatment of multiple sclerosis. ACS Chem. Biol. 2014, 9, 156−163. (5) Swedberg, J. E.; Mahatmanto, T.; Abdul Ghani, H.; de Veer, S. J.; Schroeder, C. I.; Harris, J. M.; Craik, D. J. 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. (6) Wong, C. T. T.; Rowlands, D. K.; Wong, C. H.; Lo, T. W. C.; Nguyen, G. K. T.; Li, H. Y.; Tam, J. P. Orally active peptidic bradykinin B-1 receptor antagonists engineered from a cyclotide scaffold for inflammatory pain treatment. Angew. Chem., Int. Ed. 2012, 51, 5620−5624. (7) Gilding, E. K.; Jackson, M. A.; Poth, A. G.; Henriques, S. T.; Prentis, P. J.; Mahatmanto, T.; Craik, D. J. Gene coevolution and regulation lock cyclic plant defence peptides to their targets. New Phytol. 2016, 210, 717−730. (8) Henriques, S. T.; Craik, D. J. Cyclotide structure and function: the role of membrane binding and permeation. Biochemistry 2017, 56, 669−682. (9) Gruber, C. W.; Elliott, A. G.; Ireland, D. C.; Delprete, P. G.; Dessein, S.; Göransson, U.; Trabi, M.; Wang, C. K.; Kinghorn, A. B.; Robbrecht, E.; Craik, D. J. Distribution and evolution of circular miniproteins in flowering plants. Plant Cell 2008, 20, 2471−2483. (10) Craik, D. J.; Malik, U. Cyclotide biosynthesis. Curr. Opin. Chem. Biol. 2013, 17, 546−554.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

David J. Craik: 0000-0003-0007-6796 Notes

The authors declare no competing financial interest. Biographies Simon J. de Veer is a National Health and Medical Research Council (NHMRC) Early Career Fellow at the Institute for Molecular Bioscience (IMB), located at The University of Queensland (UQ), Brisbane, Australia. He received his Ph.D. from Queensland University of Technology, and his research focuses on using naturally occurring cyclic peptides as templates for designing peptide-based tools that target enzymes and cell-surface receptors. 1563

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Accounts of Chemical Research (11) Nguyen, G. K. T.; Wang, S. J.; Qiu, Y. B.; Hemu, X.; Lian, Y. L.; Tam, J. P. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis. Nat. Chem. Biol. 2014, 10, 732−738. (12) Harris, K. S.; Durek, T.; Kaas, Q.; Poth, A. G.; Gilding, E. K.; Conlan, B. F.; Saska, I.; Daly, N. L.; van der Weerden, N. L.; Craik, D. J.; Anderson, M. A. Efficient backbone cyclization of linear peptides by a recombinant asparaginyl endopeptidase. Nat. Commun. 2015, 6, 10199. (13) Mahatmanto, T.; Mylne, J. S.; Poth, A. G.; Swedberg, J. E.; Kaas, Q.; Schaefer, H.; Craik, D. J. The evolution of Momordica cyclic peptides. Mol. Biol. Evol. 2015, 32, 392−405. (14) Göransson, U.; Burman, R.; Gunasekera, S.; Strömstedt, A. A.; Rosengren, K. J. Circular proteins from plants and fungi. J. Biol. Chem. 2012, 287, 27001−27006. (15) Weidmann, J.; Craik, D. J. Discovery, structure, function, and applications of cyclotides: Circular proteins from plants. J. Exp. Bot. 2016, 67, 4801−4812. (16) Daly, N. L.; Rosengren, K. J.; Henriques, S. T.; Craik, D. J. NMR and protein structure in drug design: Application to cyclotides and conotoxins. Eur. Biophys. J. 2011, 40, 359−370. (17) Craik, D. J. Host-defense activities of cyclotides. Toxins 2012, 4, 139−156. (18) Jagadish, K.; Camarero, J. A. Cyclotides, a promising molecular scaffold for peptide-based therapeutics. Biopolymers 2010, 94, 611− 616. (19) Poth, A. G.; Chan, L. Y.; Craik, D. J. Cyclotides as grafting frameworks for protein engineering and drug design applications. Biopolymers 2013, 100, 480−491. (20) Burman, R.; Gunasekera, S.; Stromstedt, A. A.; Göransson, U. Chemistry and biology of cyclotides: Circular plant peptides outside the box. J. Nat. Prod. 2014, 77, 724−736. (21) Henriques, S. T.; Craik, D. J. Cyclotides as templates in drug design. Drug Discovery Today 2010, 15, 57−64. (22) Craik, D. J., Ed. Advances in Botanical Research: Plant Cyclotides; Academic Press: London, 2015; Vol. 76. (23) Mulvenna, J. P.; Wang, C.; Craik, D. J. CyBase: a database of cyclic protein sequence and structure. Nucleic Acids Res. 2006, 34, D192−D194. (24) Qu, H.; Smithies, B. J.; Durek, T.; Craik, D. J. Synthesis and protein engineering applications of cyclotides. Aust. J. Chem. 2017, 70, 152−161. (25) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Synthesis of proteins by native chemical ligation. Science 1994, 266, 776. (26) Daly, N. L.; Love, S.; Alewood, P. F.; Craik, D. J. Chemical synthesis and folding pathways of large cyclic polypeptides: Studies of the cystine knot polypeptide kalata B1. Biochemistry 1999, 38, 10606− 10614. (27) Blanco-Canosa, J. B.; Dawson, P. E. An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem., Int. Ed. 2008, 47, 6851−6855. (28) Aboye, T.; Kuang, Y.; Neamati, N.; Camarero, J. A. Rapid parallel synthesis of bioactive folded cyclotides by using a tea-bag approach. ChemBioChem 2015, 16, 827−833. (29) Cheneval, O.; Schroeder, C. I.; Durek, T.; Walsh, P.; Huang, Y. H.; Liras, S.; Price, D. A.; Craik, D. J. Fmoc-based synthesis of disulfide-rich cyclic peptides. J. Org. Chem. 2014, 79, 5538−5544. (30) Simonsen, S. M.; Sando, L.; Rosengren, K. J.; Wang, C. K.; Colgrave, M. L.; Daly, N. L.; Craik, D. J. Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity. J. Biol. Chem. 2008, 283, 9805−9813. (31) Barry, D. G.; Daly, N. L.; Clark, R. J.; Sando, L.; Craik, D. J. Linearization of a naturally occurring circular protein maintains structure but eliminates hemolytic activity. Biochemistry 2003, 42, 6688−6695. (32) Henriques, S. T.; Huang, Y. H.; Rosengren, K. J.; Franquelim, H. G.; Carvalho, F. A.; Johnson, A.; Sonza, S.; Tachedjian, G.; Castanho, M. A.; Daly, N. L.; Craik, D. J. Decoding the membrane activity of the cyclotide kalata B1: the importance of phosphatidyle-

thanolamine phospholipids and lipid organization on hemolytic and anti-HIV activities. J. Biol. Chem. 2011, 286, 24231−24241. (33) Henriques, S. T.; Huang, Y. H.; Castanho, M. A.; Bagatolli, L. A.; Sonza, S.; Tachedjian, G.; Daly, N. L.; Craik, D. J. Phosphatidylethanolamine binding is a conserved feature of cyclotide-membrane interactions. J. Biol. Chem. 2012, 287, 33629−33643. (34) Grage, S. L.; Sani, M. A.; Cheneval, O.; Henriques, S. T.; Schalck, C.; Heinzmann, R.; Mylne, J. S.; Mykhailiuk, P. K.; Afonin, S.; Komarov, I. V.; Separovic, F.; Craik, D. J.; Ulrich, A. S. Orientation and location of the cyclotide kalata B1 in lipid bilayers revealed by solid-state NMR. Biophys. J. 2017, 112, 630−642. (35) Herrmann, A.; Svangård, E.; Claeson, P.; Gullbo, J.; Bohlin, L.; Göransson, U. Key role of glutamic acid for the cytotoxic activity of the cyclotide cycloviolacin O2. Cell. Mol. Life Sci. 2006, 63, 235−245. (36) Cascales, L.; Henriques, S. T.; Kerr, M. C.; Huang, Y. H.; Sweet, M. J.; Daly, N. L.; Craik, D. J. Identification and characterization of a new family of cell-penetrating peptides: cyclic cell-penetrating peptides. J. Biol. Chem. 2011, 286, 36932−36943. (37) Henriques, S. T.; Huang, Y. H.; Chaousis, S.; Sani, M. A.; Poth, A. G.; Separovic, F.; Craik, D. J. The prototypic cyclotide kalata B1 has a unique mechanism of entering cells. Chem. Biol. 2015, 22, 1087− 1097. (38) Contreras, J.; Elnagar, A. Y. O.; Hamm-Alvarez, S. F.; Camarero, J. A. Cellular uptake of cyclotide MCoTI-I follows multiple endocytic pathways. J. Controlled Release 2011, 155, 134−143. (39) Yang, R.; Wong, Y. H.; Nguyen, G. K. T.; Tam, J. P.; Lescar, J.; Wu, B. Engineering a catalytically efficient recombinant protein ligase. J. Am. Chem. Soc. 2017, 139, 5351−5358. (40) Quimbar, P.; Malik, U.; Sommerhoff, C. P.; Kaas, Q.; Chan, L. Y.; Huang, Y. H.; Grundhuber, M.; Dunse, K.; Craik, D. J.; Anderson, M. A.; Daly, N. L. High-affinity cyclic peptide matriptase inhibitors. J. Biol. Chem. 2013, 288, 13885−13896. (41) Thongyoo, P.; Bonomelli, C.; Leatherbarrow, R. J.; Tate, E. W. Potent inhibitors of beta-tryptase and human leukocyte elastase based on the MCoTI-II scaffold. J. Med. Chem. 2009, 52, 6197−6200. (42) Sommerhoff, C. P.; Avrutina, O.; Schmoldt, H. U.; GabrijelcicGeiger, D.; Diederichsen, U.; Kolmar, H. Engineered cystine knot miniproteins as potent inhibitors of human mast cell tryptase beta. J. Mol. Biol. 2010, 395, 167−175. (43) Getz, J. A.; Rice, J. J.; Daugherty, P. S. Protease-resistant peptide ligands from a knottin scaffold library. ACS Chem. Biol. 2011, 6, 837− 844. (44) 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 highaffinity inhibitors of human matriptase-1. PLoS One 2013, 8, e76956. (45) Chan, L. Y.; Craik, D. J.; Daly, N. 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. (46) Huang, Y. H.; Henriques, S. T.; Wang, C. K.; Thorstholm, L.; Daly, N. L.; Kaas, Q.; Craik, D. J. Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold. Sci. Rep. 2015, 5, 12974. (47) Getz, J. A.; Cheneval, O.; Craik, D. J.; Daugherty, P. S. Design of a cyclotide antagonist of neuropilin-1 and −2 that potently inhibits endothelial cell migration. ACS Chem. Biol. 2013, 8, 1147−1154. (48) Aboye, T. L.; Ha, H.; Majumder, S.; Christ, F.; Debyser, Z.; Shekhtman, A.; Neamati, N.; Camarero, J. A. Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity. J. Med. Chem. 2012, 55, 10729−10734. (49) Eliasen, R.; Daly, N. L.; Wulff, B. S.; Andresen, T. L.; CondeFrieboes, K. W.; Craik, D. J. Design, synthesis, structural and functional characterization of novel melanocortin agonists based on the cyclotide kalata B1. J. Biol. Chem. 2012, 287, 40493−40501. (50) Koehbach, J.; O’Brien, M.; Muttenthaler, M.; Miazzo, M.; Akcan, M.; Elliott, A. G.; Daly, N. L.; Harvey, P. J.; Arrowsmith, S.; Gunasekera, S.; Smith, T. J.; Wray, S.; Göransson, U.; Dawson, P. E.; Craik, D. J.; Freissmuth, M.; Gruber, C. W. Oxytocic plant cyclotides 1564

DOI: 10.1021/acs.accounts.7b00157 Acc. Chem. Res. 2017, 50, 1557−1565

Article

Accounts of Chemical Research as templates for peptide G protein-coupled receptor ligand design. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 21183−21188. (51) Jagadish, K.; Gould, A.; Borra, R.; Majumder, S.; Mushtaq, Z.; Shekhtman, A.; Camarero, J. A. Recombinant expression and phenotypic screening of a bioactive cyclotide against α-synucleininduced cytotoxicity in baker’s yeast. Angew. Chem., Int. Ed. 2015, 54, 8390−8394.

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DOI: 10.1021/acs.accounts.7b00157 Acc. Chem. Res. 2017, 50, 1557−1565