Discovery and Optimization of Peptide Macrocycles

Abstract

Introduction: Macrocyclic peptides are generally more resistant to proteolysis and often have

higher potency than linear peptides and so they are excellent leads in drug design. Their study is

significant because they offer the potential as a new generation of drugs that are potent and

specific, and thus might have fewer side effects than traditional small molecule drugs.

Areas covered: This article covers macrocyclic drug leads based on nature-derived cyclic peptides as well as synthetic cyclic peptides and close derivatives. The natural peptides include cyclotides, sunflower-derived peptides, theta-defensins and orbitides. Technologies to make engineered cyclic peptides covered here include cyclization via amino acid linkers, CLIPS, templates, and stapled peptides.

Expert opinion: Macrocyclic peptides are promising drug leads and several are in clinical trials.

In our opinion, they offer key advantages over traditional small molecule drugs, as well as some advantages over protein-based ‘biologics’ such as antibodies or growth factors. These include the ability to penetrate cells and attack intracellular targets such as protein-protein interactions as well as to hit extracellular targets. Some macrocyclic peptides such as cyclotides offer the potential for production in plants, thus reducing manufacture costs and potentially increasing opportunities for their distribution to developing countries at low cost.

Keywords: Cyclic peptides, cyclotides, drug design, kalata B1, peptide macrocycles, stapled peptides

Abbreviations: CCK, cyclic cystine knot; SFTI, sunflower trypsin inhibitor; MALDI-TOF,

matrix-assisted laser desorption/ionization time of flight; MCoTI, Momordica cochinchinensis

trypsin inhibitor; RTD, rhesus theta-defensin; AEP, asparaginyl endopeptidase; BTD, baboon

theta-defensin; KLK4, kallikrein-related peptidase 4; PawS, Preproalbumin with SFTI-1; PDP,

PawS derived peptides; SICLOPPS, split intein-mediated curcular ligation of peptide and proteins; MOrPHs, macrocyclic organo-peptide hybrids; CLIPS, chemical ligation of peptides

onto scaffolds; TABA, N,N′,N′′-(benzene-1,3,5-triyl)-tris(2-bromoacetamide); GHRH, Growth hormone-releasing hormone.

1. Introduction

Peptide macrocycles have stimulated much interest in the field of drug discovery due to their

unique structural and biopharmaceutical properties. Cyclization of their linear parent peptides can

be achieved via a range of connectivities, including head-to-tail, sidechain-to-sidechain or termini-

to-sidechain linkages; it is this variety that results in a structurally diverse class of molecules, both

from natural and synthetically derived sources. Macrocycles have captivated the imagination of

medicinal chemists because of their diverse design applications, including the incorporation of

bioactive motifs into cyclic peptide scaffolds, or the stabilization and optimization of bioactive

structures using synthetic linkages (Figure 1). There are currently ~30 peptide macrocycles

registered or in clinical development, with cyclosporin A the only one to be administered orally

[1].

-Insert Figure 1 here

Despite their promise, peptide macrocycles are an underrepresented class within marketed

drugs, consistent with Lipinski’s “rule-of-five” [2], which notes a preference for a molecular

weight of lower than 500 Da amongst approved drugs. Notwithstanding this size rule for drug-like

properties, many large biologics such as engineered antibodies have larger molecular weights

(>5000 Da) or violate other aspects of Lipinski’s rules. These biologics typically have the

advantage of exquisite target specificity but lack the oral bioavailability of ‘small molecule’ drugs,

thus requiring alternative administration methods such as injection. An example of a high

molecular weight blockbuster injectable drug is insulin, for the treatment of diabetes and

adalimumab for arthritis. Molecular weight alone does not preclude oral activity, and in an

extension of the Lipinski rules Veber et al. noted that, in some cases, oral bioactivity can be

achieved even for molecular weights of greater than 500 Da, provided other biophysical properties

are suitable. In particular, molecular flexibility and polar surface area are important considerations

in the development of orally bioavailable peptide macrocycles [3].

Peptide macrocycles fill a gap between small molecule drugs and biologics, with their sizes typically ranging from 500–5000 Da. They can exhibit high target specificity and, in some cases, cellular permeability and/or oral bioavailability, and their size is sufficient to target intracellular protein-protein interactions. This is important because protein-protein interfaces have previously been perceived generally as ‘undruggable’, i.e. not able to be targeted by either small molecule drugs or biologics, so the availability of technologies to address them represents a major advance in the field of drug design. This article describes recent advances in the optimization of naturally and synthetically derived peptide macrocycles in the development of leads for a range of therapeutic targets. We focus on studies published over the last few years, as earlier background

literature in this field is covered in a number of excellent reviews [4-6].

2. Natural peptide macrocycles in drug discovery

Naturally occurring peptide macrocycles are structurally diverse and have been isolated from

bacteria [7], fungi [8], plants [8] and mammals [9]. Their diversity spans a range from small pentapeptides with no disulfide bonds to large polypeptides with multiple disulfide bonds, as schematically illustrated in Figure 2. In addition to the interest generated from their structures, these peptides have been extensively investigated for their biological functions and applications in drug design, based largely on their intrinsically high stability and ability to be engineered to optimize function. In particular, certain classes of naturally occurring peptide macrocycles have been utilized as molecular scaffolds, whereby a new or modified biological function is introduced into the molecule whilst maintaining the intrinsic stability of the macrocycle. Amongst the various classes of natural peptide macrocycles, we focus here on ribosomally synthesized disulfide-rich peptides of fewer than 50 residues as these are a particularly well studied target class. Bacterial and fungal peptide macrocycles typically do not include disulfide bonds and will not be covered herein; readers are referred to other recent literature on these microorganism-derived macrocycles

[7, 8, 10].

-Insert Figure 2 here

2.1 Cyclotides

The most abundant group of ribosomally synthesized peptide macrocycles are head-to-tail cyclized peptides known as cyclotides, found predominantly in the Violaceae (violet) and Rubiaceae

(coffee) plant families [11]. The prototypic cyclotide, kalata B1, was first discovered as the active ingredient in a tea made from the plant Oldenlandia affinis, used in a traditional African medicine to accelerate childbirth. Gran isolated the bioactive peptide from O. affinis and partially characterized its amino acid content in the early 1970s [12], but it was not until some 20 years later that the macrocyclic structure was elucidated [13]. Since then, more than 360 cyclotides have been

characterized, as documented in CyBase [14], and it is likely that this number will increase, with

estimates of tens of thousands of cyclotides existing within the Rubiaceae family alone [11].

Cyclotides comprise ~30 amino acids and exhibit a unique structure known as the cyclic

cystine knot (CCK). In this structure two disulfide bonds and their connecting backbone segments

form a ring that is threaded by a third disulfide bond, with this knot motif embedded within a cyclic

peptide backbone, as illustrated in Figure 3. This conserved structural feature engenders cyclotides

with ultra-stable properties, including resistance to thermal, chemical or enzymatic breakdown

[15]. Three cyclotide subfamilies have been defined: the Möbius, bracelet and trypsin inhibitor

cyclotides. The Möbius and bracelet families differ mainly in the cis or trans configuration of an

X-Pro peptide bond in one of the six backbone ‘loops’ of the cyclotide framework [16], with kalata

B1 being the prototypic Möbius cyclotide and cycloviolacin O2 the most well studied bracelet

cyclotide. The trypsin inhibitor subfamily currently comprises eight peptides (MCoTI-I–VIII) isolated from the tropical vine Momordica cochinchinensis. Members of this subfamily have very

different sequences from Mobius or bracelet cyclotides yet maintain the CCK structure [17, 18];

of these MCoTI-I and MCoTI-II are the most well studied examples.

-Insert Figure 3 here

Natural cyclotides have a diverse range of biological activities, including anti-HIV [16,

19], anti-microbial [20], hemolytic [20-23], and cancer cell toxicity [24, 25], as well as pesticidal

activities, with the latter presumed to be their natural function [26-29]. However, they have gained most attention for their applications as molecular scaffolds in drug design. With synthetic protocols for cyclotides now well established [30-32], various groups have ‘grafted’ foreign bioactive epitopes into cyclotide scaffolds to introduce novel bioactivities. This grafting concept was first explored by Clark et al., who introduced non-native hydrophobic residues into loop 5 of kalata B1

and demonstrated the plasticity of the CCK framework to accommodate changes in the primary

structure whilst maintaining a native fold [33]. Following that study, a wide range of epitopes have

been grafted into cyclotides, including α-helices into the MCoTI-I cyclotide, demonstrating the

scaffold’s ability to accommodate diverse structures [34, 35], with the grafted sequences ranging

in size from 1-25 residues. Not all grafted epitopes lead to a stable cyclotide fold. For example, in a study directed at developing leads for the treatment of multiple sclerosis Wang et al. noted that although many grafted analogues folded satisfactorily and led to bioactive leads, certain epitopes consisting of nine or ten residues did not lead to a native kalata B1 globular conformation [36]. In

a separate study aimed at the development of analgesic cyclotides, it was noted that grafting of a

nine-residue sequence into loop 6 did form a stable globular conformation of kalata B1, suggesting

that sequence composition of the epitope has more of an influence than fragment length on folding

and stability [37]. Additionally, the choice of target loop within cyclotides has been shown to

significantly influence the stability of the grafted product [36]. Overall, cyclotides from the Mobius and trypsin inhibitor subfamilies are more amenable to correct folding using solid phase peptide synthesis than members of the bracelet subfamily. Efficient routes to the chemical synthesis of bracelet cyclotides indeed remains a great challenge.

Cyclotide grafting has now been applied to a range of disease targets, including cancer [34,

35, 38, 39], obesity [40], angiogenesis conditions [41], inflammatory pain [37], and multiple sclerosis [36, 42], amongst others. In the analgesic example noted above, Wong et al. reported the oral activity of a grafted cyclotide consisting of a nine-residue bradykinin B1 receptor antagonist inserted into loop 6 of kalata B1 [37]. That study was the first example of a synthetic orally active cyclotide, a phenomenon recently reinforced by Thell et al. [42], who reported an orally active kalata B1 variant for the treatment of multiple sclerosis. Despite the limited oral bioavailability

commonly observed for peptide-based drug leads [4], the kalata peptide was detected in serum by

MALDI-TOF two hours after oral administration in mice, and had no detrimental effects on the

animals, suggestive of a good safety profile [42]. These studies support the idea that cyclotide

scaffolds can be used as orally active drugs capable of withstanding the enzymatic and pH

conditions of the gastrointestinal tract without having toxic side effects.

Certain native cyclotide scaffolds have the ability to translocate across cellular membranes,

thus offering the potential to target intracellular protein-protein interactions. MCoTI-II was the

first cyclotide identified as having cell-penetrating properties [43], a finding that was later also

demonstrated for kalata B1 [44] and MCoTI-I [45], leading to the classification of a new family of molecules called ‘cyclic cell-penetrating peptides’ [44]. Interestingly, different cyclotide subfamily members have different modes of cellular internalization: the trypsin inhibitor cyclotide

MCoTI-II appears to penetrate cells via macropinocytosis [44], whereas the Mobius cyclotide kalata B1 enters cells via both endocytosis and direct cellular translocation [46]. Detailed studies on the MCoTI-II framework have suggested that permeability can be significantly enhanced by increasing the overall positive charge of the scaffold [47]. This finding is consistent with a grafting study by Huang et al. demonstrating increased permeability of a positively charged MCoTI-II scaffold in the design of substrate-based BCR-ABL kinase inhibitors with potential for the treatment of chronic myeloid leukemia [38]. Overall, it is still fair to say that the structural parameters that influence the cellular internalization of cyclotides are not yet thoroughly understood.

A very new development in this field has been the demonstration of enzyme-medicated cyclisation of peptide macrocycles. The discovery and characterization of asparaginyl enzymes involved in the biosynthesis of cyclotides has in particular facilitated the ability to make linear

peptides using either solid phase peptide synthesis or recombinant methods and then cyclize them

using asparaginyl endopeptidase (AEP). This has been demonstrated for the AEP butelase-1

isolated from Butterfly pea [48] and for a recombinant form of an AEP from O. affinis [49].

2.2 θ-Defensins

θ-Defensins are head-to-tail cyclic peptides originally discovered in the leucocytes of rhesus

monkeys [50]. They comprise ~18 amino acids and have three parallel disulfide bonds forming an

arrangement known as the cyclic cystine ladder [51]. Much like the cyclotide scaffold, their

disulfide connectivity engenders them with exceptional thermal and enzymatic stability [52]. To

date, 11 θ-defensins have been isolated, including six from rhesus monkeys (RTD-1 to RTD-6),

and five baboon θ-defensins (BTD1-5) from olive and hamadryas baboons [53]. θ-Defensins are

implicated in the innate immunity of Old World monkeys, and RTD-1 accounts for around 50%

of the total θ-defensin content in these primates [54]. Several studies have investigated RTD-1 and

related θ-defensins for antimicrobial [55-57] and antiviral activities [58], as well as for insulin resistance properties [59]. Importantly, θ-defensins display low toxicity and minimal immunogenicity in murine models [55, 58, 59].

Six θ-defensin pseudogenes have been identified in the human genome, and all contain a premature stop codon upstream of the precursor signal peptide, which inhibits translation.

Interestingly, chemical synthesis of the encoded sequences has led to the development of synthetic

θ-defensins termed retrocyclins [60]. Retrocyclins inhibit replication of HIV-1 [60], which has led

to speculation as to whether the evolutionary conversion of the genes encoding retrocyclins into

pseudogenes has contributed to our susceptibility to HIV infection [61].

θ-Defensins are interesting molecules for drug design because of their antimicrobial and

other biological activities in combination with excellent thermal and proteolytic stability, and low

toxicity [62]. Furthermore, the development of efficient synthetic protocols [51, 52, 63] has

prompted the investigation of θ-defensins as molecular scaffolds. This application was first

demonstrated with an integrin-binding sequence (Arg-Gly-Asp) motif grafted into the hairpin

loops of RTD-1. The resulting analogue had potent binding to integrin (IC50 18 nM) and had high

serum stability, confirming the value of θ-defensins as functional scaffolds [62]. Conibear et al.

also demonstrated stabilization of anticancer epitopes using the baboon θ-defensin BTD-2 as a scaffold [64]. As more grafting examples become available, we will gain a greater understanding of the plasticity of the θ-defensins as molecular scaffolds for drug discovery.

2.3 PawS-Derived Peptides

These cyclic peptides are encoded by the PawS1 (Preproalbumin with SFTI-1) gene and were first

discovered in the common sunflower (Helianthus annuus). The family comprises ~20

characterized peptides, which are referred to as sunflower trypsin inhibitors (SFTI) or ‘PawS-

derived peptides’ (PDPs) [65, 66]. The most well-known of these peptides is SFTI-1, a 14 residue

cyclic peptide that incorporates two antiparallel β-strands stabilized by a single disulfide bond

[65]. SFTI-1 has sequence homology with a class of peptides known as the Bowman Birk

inhibitors and has been investigated for its anti-inflammatory and anti-cancer properties [67].

SFTI-1 is a stable molecule due to the combination of its cyclic backbone, disulfide bond and

hydrogen bond network [68]. This stability has resulted in its wide application as a molecular

scaffold in drug design [5, 69]. Like cyclotides, SFTI-1 has been reported to have cell penetrating properties [44] but the mechanism of translocation is different to, and weaker than, that of its cyclotide relatives and probably occurs via non-specific endocytosis-dependent cellular uptake

[47].

Recent grafting studies investigated the SFTI-1 framework for the incorporation of anti- angiogenic activity [70], pro-angiogenic activity [41], anti-fibril activity [71] and a variety of protease inhibition activities [72-74]. One such example was the incorporation of small pro- angiogenic peptide sequences into the SFTI-1 scaffold to develop molecules for therapeutic

angiogenesis. The most potent peptide incorporated a heptapeptide sequence from osteopontin

(Ser-Val-Val-Tyr-Gly-Leu-Arg), which induced angiogenesis at nanomolar concentrations in

mice [41]. SFTI-1 analogues have also been used in various cancer targeting applications. For

example, Swedberg et al. used this scaffold to target kallikrein-related peptidase 4 (KLK4)

inhibition, whereby a tetrapeptide sequence (Phe-Cys-Gln-Arg) was incorporated into SFTI-1 and

led to a potent and selective KLK4 inhibitor [75], with potential for the treatment of prostate

cancer. Another recent cancer-related study reported the grafting of a thrombospondin-1 mimetic

heptapeptide fragment (Gly-Val-Ile-Thr-Arg-Ile-Arg) into SFTI-1, resulting in a stable, non-toxic,

potent anti-angiogenesis peptide [70].

2.4 Orbitides

These disulfide-less cyclic peptides comprise 5–12 residues and are found in a variety of plants

from flax and related families. Overall, this class has not yet gained much attention in drug design,

but a few examples such as the cyclolinopeptides or curcacyclines, have been reported to have

anticancer [76] or immunosuppressive properties [77, 78]. For example, cyclolinopeptide A has

immunosuppressive activity believed to be mediated through its binding to cyclophilin A, similar

to that of the gold standard immunosuppressive cyclosporin A [77]. Their small size, similar to

many synthetic peptides routinely made by pharmaceutical or medicinal chemists, makes them

synthetically accessible useful frameworks for mimicking turn regions of proteins. We anticipate

that applications of this class of natural products will gain increasing prominence over the next

few years.

3. Synthetic macrocyclization of peptides in drug discovery

Synthetic macrocyclization is a strategy that has been widely used to overcome some of

the unfavorable biopharmaceutical properties of linear peptides. Two main approaches are the

stabilization of bioactive peptides using amino acid linkers to bridge the termini, and the

stabilization of peptide epitopes using non-native chemical entities such as small molecule linkers

or non-proteinogenic amino acids (Figure 4). These strategies have led to the development of a range of macrocycles that are gaining considerable attention because of their favorable biopharmaceutical properties. Here we discuss various types of synthetic macrocyclization approaches, including those leading to head-to-tail cyclized peptides, small organic molecule stabilized peptides, stapled peptides and β-hairpin mimetics. Several other macrocyclization approaches based on genetically encoded peptides have been applied, including the split intein- mediated circular ligation of peptide and proteins (SICLOPPS) [79]; macrocyclic organo-peptide hybrids (MOrPHs) [80]; and the ribosomal synthesis of nonstandard peptide macrocycles using genetic code reprogramming technology [81]. Space limitation prevents a full discussion of these here, but they have been the topic of a number of excellent recent reviews [80-82].

-Insert Figure 4 here

3.1 Head-to-tail cyclization of bioactive disulfide-rich peptides

Head-to-tail cyclization is a broadly applicable strategy to stabilize peptides. Amongst disulfide- rich peptides, head-to-tail cyclization has had particular application in conotoxins, which are

peptides from the venoms of marine cone snails that have a wide range of ion-channel targets and

hence are of therapeutic interest. The first exemplified cyclization of a conotoxin involved the

stabilization of the model α-conotoxin MII using 5-7 amino acid linkers [83]. A later study

introduced a six-residue linker (Gly-Gly-Ala-Ala-Gly-Gly) into the analgesic α-conotoxin Vc.1.1, resulting in a head-to-tail cyclized peptide (Figure 4a) that exhibited enhanced activity over the acyclic precursor and had oral activity in a rat model of neuropathic pain. Indeed, the cyclic Vc1.1 was significantly more potent than the gold standard neuropathic pain drug gabapentin [84]. Many studies have since applied cyclization to other conotoxins, including α-RgIA [85], α-ImI [86], α-

AuIB [87, 88], χ-MrIA [89], ω-MVIIA [90], as well as to the P-superfamily conotoxins gm9a and bru9a [91]. In almost all cases, the cyclic peptides display increased stability compared to their acyclic counterpart. The typical close proximity of the N and C termini make conotoxins excellent

targets for head-to-tail cyclization and subsequent pharmaceutical improvement.

Head-to-tail cyclization has also been applied to a variety of other venom-derived peptides.

For example, Chan et al. engineered the Acanthoscurria gomesiana spider peptide gomesin to incorporate a linker between the N and C termini of the native peptide [92]. Cyclization led to increased selective cytotoxicity to a cancer cell line, improved antimalarial activity and a slight improvement in stability. On the other hand, cyclization of the sea anemone peptide APETx2 [93] and the iron regulatory peptide hormone hepcidin [94] resulted in improved peptide stability, albeit with the loss of bioactivity. In these cases, the loss of bioactivity was probably due to the disruption of interactions of the N or C termini with their respective binding targets. A similar result was

recently reported for the cyclisation of the conotoxin PVIIA [95, 96]. Overall, head-to-tail

cyclization of bioactive peptides has proven to be an effective tool for improving stability, but

careful design of peptide linker is required to maintain biological activity.

3.2 Small organic molecule stabilization of peptide epitopes

Small organic molecules have been widely used to constrain peptide epitopes for the mimicry of

protein surfaces. One technology commonly employed in the synthesis of these peptides is known

as the chemical ligation of peptides onto scaffolds (CLIPS), with the products commonly referred

to as CLIPS-constrained peptides. CLIPS linkers are small organic ligands generally comprised of

poly(bromomethyl)benzene complexes that can be efficiently bound to cysteine residues under

mild reaction conditions in good yield (~80%) [97]. These linkers can be designed to incorporate a single, double or triple peptide loop with the incorporation of two, three or four cysteine residues,

respectively [97] (Figure 4b). This type of linker has been show to dramatically influence the

conformational preferences of the peptides, resulting in significant variation of the activity [98].

For example, Chen et al. showed that the linker N,N′,N′′-(benzene-1,3,5-triyl)-tris(2-

bromoacetamide) can form non-covalent interactions with the constructed peptide that further

stabilizes it and potentially increases target binding affinities [99].

The use of small organic peptide-stabilizing molecules has been demonstrated in a range

of drug design applications. In particular, these molecules can be applied for use in phage-encoded

combinatorial libraries due to their synthesis being achievable under mild reaction conditions

[100]. For example, Heinis et al. employed phage-encoded libraries with the use of CLIPS to

inhibit human plasma kallikrein at nanomolar affinity [101, 102]. Using similar methods a range

of bicyclic peptides has been developed to explore novel therapeutic targets such as coagulation

factor XIIa [103] as well ligands that can effectively bind to β-catenin [104]. Pharmacological

studies of this class of molecules has found that they can be non-covalently conjugated to serum

albumin to successfully ‘piggyback’ them into solid tumors [105]. Furthermore, some have cell-

penetrating properties that can be optimized. For example, certain bicyclic peptides were demonstrated to have increased cellular permeability by introducing arginine-rich sequences into the peptidic component of the molecules [106].

3.3 Stapled peptides

α-Helices are commonly implicated in protein-protein interactions that have potential as therapeutic targets, and small helical motifs can be used to block these protein-protein interactions.

Utilizing such structural motifs requires stabilization of the helix, which can be achieved through synthetically interlinking proximate amino acid sidechains on one side of the helix. This approach has led to a class of peptides known as stapled peptides, which were first developed by Verdine and colleagues who stapled two non-natural amino acids in a helix with a hydrocarbon linker to enhance helicity and metabolic stability [107]. Stapled peptides are a structurally diverse class of molecules that can involve links through residues i to i+3, i to i+4, or i, i+7 (Figure 5a). Moreover, staples can comprise disulfide, thioester, diester, lactam, triazole, or pyrazole linkages, but hydrocarbon linkages are most commonly used [6]. Hydrocarbon linkages are formed via ring- closing metathesis of α,α-disubstituted non-natural amino acids bearing olefin tethers [107].

Stapled peptides have been used to target a range of diseases that involve either intracellular or extracellular targets. At the forefront of this class of molecules is the orally bioavailable p53-

MDM2/MDMX agonist ALRN-6924, and the growth-hormone-releasing hormone (GHRH) agonist ALRN-5281, both of which have progressed to clinical development [108, 109].

A desirable feature of stapled peptides is the ability to penetrate cells [110-113]. Such stapled peptides have been developed for a wide variety of intracellular targets, including BCL-2 family proteins [110, 114, 115], MDM2/MDMX [116-118], HIV-1 integrase [119], and estrogen receptors [120]. Stapled peptides have been suggested to penetrate cells via a clathrin- and caveolin-independent endocytosis pathway mediated through cell surface proteoglycans, and also via a novel uncharacterized pathway [113]. Structural parameters underlying cellular internalization are generally associated with a net positive charge (+1 to +7), but a formal charge greater than +7 has been associated with significantly decreased cellular uptake [113].

Hydrophobicity is another important biophysical property of stapled peptides. Hydrocarbon staples located at the amphipathic boundary increase the hydrophobic surface area and thus can significantly enhance cellular permeability [121]. Studies on the cell-penetrating properties of stapled peptides have been the subject of scrutiny due to variations in the methods used to evaluate the peptides. For example, one study reported the stapled peptide SAHp53-8 to be an antagonist of MDM2/MDMX proteins [116], but later studies failed to replicate the activity and reported no intracellular activity of the stapled peptide [111, 117]. The development of robust and universal cellular uptake assays will go a long way to increasing our understanding of the cell-penetrating properties of stapled peptides, and indeed of peptide macrocycles in general.

-Insert Figure 5 here

3.4 β-Hairpin mimetics

Like α-helices, β-hairpins are found at the active sites of a variety of protein-protein interactions, making them idea candidates for drug leads. Many β-hairpin mimetics have been designed based on naturally occurring β-hairpin motifs from larger proteins. Typically, these molecules are replicated synthetically and cyclized via the use of a β-turn template that can be derived from

almost any β-turn-inducing motif. Moreover, they can be customized to the desired shape and size

of the β-hairpin (Figure 5b) [122, 123]. One of the most useful β-hairpin templates is the D-Pro-L-

Pro construct, which induces a type II β-turn conformation and has been successfully implemented

in the stabilization of many hairpin mimetics into defined conformations [124, 125]. The versatile

application and relative ease of synthesis of these molecules makes them excellent candidates in

drug design.

β-Hairpin mimetics have been of particular interest in the development of new

antimicrobial peptides. For example, the Robinson group and Polyphor Ltd. have developed

antimicrobial cyclic peptides inspired by the natural antimicrobial peptide protegrin-1 using the D-

Pro-L-Pro template linker. Subsequent optimization led to the development of POL7080, shown to have a novel mode of action in targeting the outer membrane β-barrel protein LptD (Imp/OstA),

which is involved in the biogenesis of lipopolysaccharide of Gram-negative bacteria [126]. The

specific binding mode of β-hairpin mimetics to LptD is not yet well understood but N-methylation

scans have implicated hydrogen bonding of Trp, Ala, and Orn at specific positions for

antimicrobial activity in a series of 14-residue β-hairpin mimetic analogues [127]. POL7080 is

currently in Phase 2 clinical trials for the treatment of ventilator-acquired pneumonia caused by P.

aeruginosa. Another cyclic β-hairpin mimetic, JB-95 (cyclo[Trp-Arg-Ile-Arg-Ile-D-Arg-Glu-Lys-

Arg-Leu-Arg-Arg-D-Pro-Pro]) has specific activity against E. coli, also targeting β-barrel proteins

in the outer membrane [128]. These examples highlight the application of β-hairpin mimetics in a

field desperate for the development of novel pharmaceuticals to overcome bacterial resistance.

In addition to being useful for antimicrobial activities, β-hairpin mimetics can be designed for a diverse range of applications. Cyclic β-hairpin analogues with potent activity have been developed to replicate the α-helix sidechain positions of the amino acids involved in the p53-

HDM2 protein-protein interaction [129, 130]. Other applications of β-hairpin mimetics include the design of a neutrophil elastase inhibitor (POL6014) inspired by the β-hairpin motif from SFTI-1, and a chemokine receptor CXCR4 antagonist (POL6326) from the horseshoe crab peptide polyphemusin II [123]. Both are currently in clinical development, highlighting the broad application of β-hairpin mimetics.

4. Small synthetic cyclic peptides as probes into oral bioavailability

We have highlighted a few examples of lead peptides with membrane permeabilizing properties,

but in general it is fair to say that the rational design of membrane-permeable peptides is not well

understood. So far, the natural product cyclosporin A is the only marketed peptide drug that is

administered orally for systemic delivery and it has a reported oral bioavailability of only around

20% [131]. Cyclosporin A comprises 11 residues, including seven N-methylated residues and a

single D-amino acid. Its oral bioavailability has been attributed to the reduction of the number of hydrogen-bond donors through N-methylation, the minimization of water solvation through shielding of polar atoms with hydrophobic sidechains and the formation of internal hydrogen bonding [132]. These properties have inspired research into other small cyclic peptides that also

incorporate non-proteinogenic amino acids. Such molecules have been used to further investigate

the biophysical properties of peptides in the hope of applying the principles in rational drug design.

In particular, a number of small cyclic peptides have been developed to further understand the conformational role of N-methylation and membrane permeability. N-methylation has been

speculated to increase permeability by allowing intermolecular hydrogen bonding with the

membrane-associated state through a conformational change from the water to membrane

environment [133]. This theory led White et al. to systematically investigate N-methylated variants

of leucine-rich hexapeptides. A resulting peptide incorporating two D-amino acids used in the

formation of two opposing β-turns (cyclo[Leu-NMe-D-Leu-NMe-Leu-Leu-D-Pro-NMe-Tyr]) achieved an oral bioavailability of 28% in mice. This demonstrated the use of N-methylated residues to induce shielding of polar amides from solvation for the development of orally bioavailable peptides [133].

Hexa-alanine constructs have also been used to investigate membrane permeability. Beck et al. used a library of 54 cyclo(D-Ala-Ala5) peptides to explore the effects of various N-

methylation patterns on cellular permeability. That study demonstrated that multiple N-

methylations improved permeability, in particular N-methylation of either two or four residues

displayed the highest permeability of all analogues [134]. Further investigations of the hexa- alanine peptides demonstrated the importance N-methylated cis-peptide bonds for permeability, another structural feature which is present in cyclosporin A [135]. These structural features of the peptide backbone may be applied to bioactive pharmacophores for the development of orally bioavailable therapeutics [134, 135].

In addition to N-methylated amino acids, other non-proteinogenic amino acids have been employed to optimize membrane permeability of small cyclic peptides. Hill and coworkers explored the role of hydrophobic side-chains as solvent shields in penta- and hexa-leucine peptides without N-methylation. They developed three orally bioavailable analogues, with the best analogue (cyclo[Leu]6) achieving 17% oral bioavailability [131]. Another study applying this principle to the heptapeptide natural product, sanguinamide A, used a branched amino acid (tert- butyl glycine) to shield the two H-bonds and polar atoms. The resulting analogue improved oral bioavailability, from 7% to 51%, in mice [132]. Solvent shielding can also be achieved by increasing rigidity in cyclic hexapeptides, as exemplified by the incorporation D-prolines to reduce

the polar surface area and thus significantly increase membrane permeability [136]. These findings

indicate that both rigidity and flexibility may be used as principles to develop membrane-

permeable cyclic peptides. Other studies on permeability suggest extended polyketide-derived γ-

amino acids can be used to increase backbone diversity whilst preserving permeability [137], but

attempts to use β-branching to increase permeability have thus far failed [138]. Overall, these

modifications may be utilized to guide the rational design of membrane-permeable peptides.

5. Conclusions

Peptide macrocycles are a diverse class of molecules that are gaining increasing attention as

prospective pharmaceuticals. Natural cyclic peptides have proven to be ultra-stable molecules that can be utilized as scaffolds to stabilize and adopt the biological properties of a foreign epitope.

This approach has now been demonstrated in applications for both intracellular and extracellular targets, and for a wide range of therapeutic applications, including cancer, cardiovascular disease, and inflammatory/infectious disease. An array of macrocyclization techniques have led to the development of several classes of peptide macrocycles, including head-to-tail cycles, CLIPS, α- helix staples and β-epitope mimetics. These classes have been shown to successfully enhance the biopharmaceutical properties of peptide macrocycles, with the most promising leads now in Phase

1 and 2 clinical trials.

6. Expert Opinion

The field of peptide macrocycles is poised at a very exciting stage, with multiple approaches being progressed and several showing great promise. At this stage, to judge whether one approach is favored over the others is premature, but several of the approaches include examples of molecules that are in advanced clinical trials. In our opinion, it is reasonable to suggest that one or more of

these molecules will make it into the market. Only if and when that happens can the field of peptide macrocycles be considered to have matured from promise to reality. The major findings in the field

so far are that peptide macromolecules are highly stable and address new target space. In particular, excitement in the field derives largely from the fact that peptide macrocycles have the potential to fill a gap in the pharmaceutical market in the 500–5000 Da molecular weight range with molecules that, in some cases, can penetrate cells to attack intracellular targets. Such targeting provides an advantage over antibodies or other large biologics which cannot penetrate cells. Furthermore, the larger size of peptide macrocycles compared to small molecule drugs gives them the advantage of being able to disrupt protein-protein interactions. Thus, peptide macromolecules open up brand new target spaces on two frontiers.

One of the weaknesses of macrocycle technology though, is that there is still no general solution to the problem of achieving high oral bioavailability. The ultimate goal in this field would be to have a greater understanding of the factors affecting oral bioavailability and to be able to build oral bioavailability into classes of therapeutics where this delivery route is desirable. To achieve this goal additional research is needed into how various classes of macrocycles cross membrane barriers and this is an active topic in the literature at present. Such studies need to address both the role of direct macrocycle-membrane interactions and also the potential role of transporter molecules. We believe that the later area is understudied and will be of particular interest over the next few years. There also remain significant synthetic challenges for some classes of macrocycles, both in terms of improving efficiency and lowering cost, and in the case of bracelet cyclotides even just developing a method that generally allows their synthesis would be a big breakthrough.

In our opinion no single modality will dominate the field of peptide macrocycles, and it

seems likely that the various modalities will be applied on a case-by-case basis and tailored for specific applications. For example, stapled peptides have particular advantages when helical epitopes are involved, whereas other templating approaches are more suitable for β-strand or turn epitopes. On the other hand, one of the ‘blue sky’ advantages of natural peptide-based macrocycles, such as cyclotides or SFTI-related peptides, is that they can in principle be incorporated into plant-based production systems. Since plants produce these molecules naturally in high yield, it seems plausible to use crop plants to produce pharmaceutically modified cyclotides, for example. This production route has the potential to reduce the cost of goods, a factor that is sometimes considered expensive for synthetically produced peptides. Furthermore, production of peptide-based pharmaceuticals in seeds could provide opportunities for the inexpensive distribution of next-generation medicines to developing countries. Of course there are many regulatory, commercial and ethical considerations that need to be addressed before such a

‘drugs in plants’ distribution scheme is enabled. In our opinion, the benefits of such an approach justify serious consideration of these issues. Irrespective of future production methods, in our opinion the potential benefits of peptide macrocycles as pharmaceuticals is now clear, and the future will hopefully see this class of molecules develop as therapeutics.

Acknowledgements

Work in our laboratory on peptide macrocycles is funded by grants from the Australian Research

Council (ARC; DP150100443), the National Health and Medical Research Council

(APP1084965), and a GSK Award for Research Excellence. DJC is an ARC Australian Laureate

Fellow (FL150100146). AMW is grateful for financial support from an Australian Postgraduate

Award and the E.M.A and M.C. Henker Postgraduate Medical Research Scholarship. We thank

Ashley Cooper for proof reading the manuscript.

Article highlights

• Naturally occurring cyclic peptides are ultra-stable and can be used as scaffolds for the

delivery of introduced bioactive epitopes.

• Peptide macrocyclization is an effective strategy to enhance the pharmacological

properties of bioactive peptides.

• Several alternative and complementary technologies exist to make peptide macrocycles.

• Some peptide macrocycles have the ability to penetrate cells to inhibit intracellular

protein-protein interactions.

• The factors underlying the membrane-penetration mechanism(s) of peptides are still not

well understood, but are being actively investigated.

• Several peptide macrocycles are now in Phase 1 and 2 clinical development.

Figure 1

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Figure 3

Figure 4

Figure 5

Bibliography

1. Giordanetto F and Kihlberg J. Macrocyclic drugs and clinical candidates: What can

medicinal chemists learn from their properties? J Med Chem. 2014;57:278-295.

doi:10.1021/jm400887j

2. Lipinski CA. Drug-like properties and the causes of poor solubility and poor

permeability. J Pharmacol Toxicol Methods. 2000;44:235-249. doi:10.1016/S1056-

8719(00)00107-6

*A paper defining five key rules used in medicinal chemistry to guide the

development of orally active small molecule drugs.

3. Veber DF, Johnson SR, Cheng HY, et al. Molecular properties that influence the oral

bioavailability of drug candidates. J Med Chem. 2002;45:2615-2623.

doi:10.1021/jm020017n

*A paper defining the molecular properties that influence oral bioavailability

independent of molecule size.

4. Craik DJ, Fairlie DP, Liras S, and Price D. The future of peptide‐based drugs. Chem Biol

Drug Des. 2013;81:136-147. doi:10.1111/cbdd.12055

*A review highlighting the challenges and future directions of peptides as drugs.

5. Northfield SE, Wang CK, Schroeder CI, et al. Disulfide-rich macrocyclic peptides as

templates in drug design. Euro J Med Chem. 2014;77:248-257.

doi:10.1016/j.ejmech.2014.03.011

6. Sawyer TK, Guerlavais V, Darlak K, and Feyfant E. Macrocyclic α-helical peptide drug

discovery, in Macrocycles in Drug Discovery. London: RSC Drug Discovery Series,

2015

7. Montalban-Lopez M, Sanchez-Hidalgo M, Cebrian R, and Maqueda M. Discovering the

bacterial circular proteins: Bacteriocins, cyanobactins, and pilins. J Biol Chem.

2012;287:27007-27013. doi:10.1074/jbc.R112.354688

8. Göransson U, Burman R, Gunasekera S, et al. Circular proteins from plants and fungi. J

Biol Chem. 2012;287:27001-27006. doi:10.1074/jbc.R111.300129

9. Lehrer RI, Cole AM, and Selsted ME. θ-Defensins: Cyclic peptides with endless

potential. J Biol Chem. 2012;287:27014-27019. doi:10.1074/jbc.R112.346098

10. Luo H, Hong SY, Sgambelluri RM, et al. Peptide macrocyclization catalyzed by a prolyl

oligopeptidase involved in alpha-amanitin biosynthesis. Chem Biol. 2014;21:1610-1617.

doi:10.1016/j.chembiol.2014.10.015

11. Gruber CW, Elliott AG, Ireland DC, et al. Distribution and evolution of circular

miniproteins in flowering plants. Plant Cell. 2008;20:2471-2483.

doi:10.1105/tpc.108.062331

12. Sletten K and Gran L. Some molecular properties of kalatapeptide B-1. A uterotonic

polypeptide isolated from Oldenlandia affinis DC. Medd Nor Farm Selsk. 1973;7:69-82.

13. Craik DJ, Daly NL, Bond T, and 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. doi:10.1006/jmbi.1999.3383

*The original paper defining the cyclotide family of peptides

14. Wang CKL, Quentin K, Chiche L, and Craik DJ. CyBase: A database of cyclic protein

sequences and structures, with application in protein discovery and engineering. Nucleic

Acids Res. 2008;36:D206-210. doi:10.1093/nar/gkm953

15. Colgrave ML and Craik DJ. Thermal, chemical, and enzymatic stability of the cyclotide

Kalata B1: The importance of the cyclic cystine knot. Biochemistry. 2004;43:5965-5975.

doi:10.1021/bi049711q

16. Daly NL, Gustafson KR, and Craik DJ. The role of the cyclic peptide backbone in the

anti-HIV activity of the cyclotide kalata B1. FEBS Lett. 2004;574:69-72.

doi:10.1016/j.febslet.2004.08.007

17. Felizmenio-Quimio ME, Daly NL, and Craik DJ. Circular proteins in plants: Solution

structure of a novel macrocyclic trypsin inhibitor from Momordica cochinchinensis. J

Biol Chem. 2001;276:22875-22882. doi:10.1074/jbc.M101666200

18. Chiche L, Heitz A, Gelly JC, et al. Squash inhibitors: From structural motifs to

macrocyclic knottins. Curr Protein Pept Sci. 2004;5:341-349.

doi:10.2174/1389203043379477

19. Gustafson KR, Walton LK, Sowder RC, Jr., et al. New circulin macrocyclic polypeptides

from Chassalia parvifolia. J Nat Prod. 2000;63:176-178. doi:10.1021/np990432r

20. Tam JP, Lu YA, Yang JL, and Chiu KW. An unusual structural motif of antimicrobial

peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc Natl Acad

Sci USA. 1999;96:8913-8918. doi:10.1073/pnas.96.16.8913

21. Ireland DC, Colgrave ML, and Craik DJ. A novel suite of cyclotides from Viola odorata:

Sequence variation and the implications for structure, function and stability. Biochem J.

2006;400:1-12. doi:10.1042/bj20060627

22. Plan MR, Göransson U, Clark RJ, et al. The cyclotide fingerprint in oldenlandia affinis:

Elucidation of chemically modified, linear and novel macrocyclic peptides.

ChemBioChem. 2007;8:1001-1011. doi:10.1002/cbic.200700097

23. Simonsen SM, Sando L, Rosengren KJ, et al. Alanine scanning mutagenesis of the

prototypic cyclotide reveals a cluster of residues essential for bioactivity. J Biol Chem.

2008;283:9805-9813. doi:10.1074/jbc.M709303200

24. Lindholm P, Göransson U, Johansson S, et al. Cyclotides: A novel type of cytotoxic

agents. Mol Cancer Ther. 2002;1:365-369.

25. Gerlach SL, Rathinakumar R, Chakravarty G, et al. Anticancer and chemosensitizing

abilities of cycloviolacin 02 from Viola odorata and psyle cyclotides from Psychotria

leptothyrsa. Biopolymers. 2010;94:617-625. doi:10.1002/bip.21435

26. Jennings C, West J, Waine C, et al. Biosynthesis and insecticidal properties of plant

cyclotides: the cyclic knotted proteins from Oldenlandia affinis. Proc Natl Acad Sci USA.

2001;98:10614-10619. doi:10.1073/pnas.191366898

27. Colgrave ML, Kotze AC, Ireland DC, et al. The anthelmintic activity of the cyclotides:

natural variants with enhanced activity. ChemBioChem. 2008;9:1939-1945.

doi:10.1002/cbic.200800174

28. Plan MR, Saska I, Cagauan AG, and Craik DJ. Backbone cyclised peptides from plants

show molluscicidal activity against the rice pest Pomacea canaliculata (golden apple

snail). J Agric Food Chem. 2008;56:5237-5241. doi:10.1021/jf800302f

29. Barbeta BL, Marshall AT, Gillon AD, et al. Plant cyclotides disrupt epithelial cells in the

midgut of lepidopteran larvae. Proc Natl Acad Sci USA. 2008;105:1221-1225.

doi:10.1073/pnas.0710338104

30. Daly NL, Clark RJ, and Craik DJ. Disulfide folding pathways of cystine knot proteins.

Tying the knot within the circular backbone of the cyclotides. J Biol Chem.

2003;278:6314-6322. doi:10.1074/jbc.M210492200

31. Göransson U and Craik DJ. Disulfide mapping of the cyclotide kalata B1. Chemical

proof of the cystic cystine knot motif. J Biol Chem. 2003;278:48188-48196.

doi:10.1074/jbc.M308771200

32. Cheneval O, Schroeder CI, Durek T, et al. Fmoc-based synthesis of disulfide-rich cyclic

peptides. J Org Chem. 2014;79:5538-5544. doi:10.1021/jo500699m

**A paper outlining a protocol for the efficient synthesis of disulfide rich cyclic

peptides.

33. Clark RJ, Daly NL, and Craik DJ. Structural plasticity of the cyclic-cystine-knot

framework: Implications for biological activity and drug design. Biochem J.

2006;394:85-93. doi:10.1042/BJ20051691

34. Ji Y, Majumder S, Millard M, et al. In vivo activation of the p53 tumor suppressor

pathway by an engineered cyclotide. J Am Chem Soc. 2013;135:11623-11633.

doi:10.1021/ja405108p

**A paper reporting the development of a stable engineered cyclotide that

antagonizes the intracellular p53 tumor suppressor pathway with nanomolar

affinity, both in vitro and in vivo.

35. D’Souza C, Henriques ST, Wang CK, et al. Using the MCoTI-II cyclotide scaffold to

design a stable cyclic peptide antagonist of SET, a protein overexpressed in human

cancer. Biochemistry. 2016;55:396-405. doi:10.1021/acs.biochem.5b00529

36. Wang CK, Gruber CW, Cemazar M, et al. Molecular grafting onto a stable framework

yields novel cyclic peptides for the treatment of multiple sclerosis. ACS Chem Biol.

2014;9:156-163. doi:10.1021/cb400548s

*A paper describing the stabilization of a myelin oligodendrocyte glycoprotein

epitope grafted into a cyclotide and the subsequent inhibition of multiple sclerosis in

mice.

37. Wong CTT, Rowlands DK, Wong CH, et al. Orally active peptidic bradykinin B1

receptor antagonists engineered from a cyclotide scaffold for inflammatory pain

treatment. Angew Chem Int Ed. 2012;51:5620-5624. doi:10.1002/anie.201200984

38. Huang YH, Henriques ST, Wang CK, et al. Design of substrate-based BCR-ABL kinase

inhibitors using the cyclotide scaffold. Sci Rep. 2015;5:12974. doi:10.1038/srep12974

*A paper reporting the design of an engineered cyclotide with inhibitory activity of

BCR-ABL tyrosine kinase for the treatment of chronic myeloid leukemia.

39. Aboye T, Meeks CJ, Majumder S, et al. Design of a MCoTI-based cyclotide with

angiotensin (1-7)-like activity. Molecules. 2016;21. doi:10.3390/molecules21020152

40. Eliasen R, Daly NL, Wulff BS, et al. Design, synthesis, structural and functional

characterization of novel melanocortin agonists based on the cyclotide kalata B1. J Biol

Chem. 2012;287:40493-40501. doi:10.1074/jbc.M112.395442

41. Chan LY, Gunasekera S, Henriques ST, et al. Engineering pro-angiogenic peptides using

stable, disulfide-rich cyclic scaffolds. Blood. 2011;118:6709-6717. doi:10.1182/blood-

2011-06-359141

42. Thell K, Hellinger R, Sahin E, et al. Oral activity of a nature-derived cyclic peptide for

the treatment of multiple sclerosis. Proc Natl Acad Sci USA. 2016;113:3960-3965.

doi:10.1073/pnas.1519960113

**A study demonstrating the oral activity of an engineered cyclotide that silences T-

cell proliferation as an immunosuppressive molecule for the treatment of multiple

sclerosis.

43. Greenwood KP, Daly NL, Brown DL, et al. The cyclic cystine knot miniprotein MCoTI-

II is internalized into cells by macropinocytosis. Int J Biochem Cell Biol. 2007;39:2252-

2264. doi:10.1016/j.biocel.2007.06.016

44. Cascales L, Henriques ST, Kerr MC, et al. Identification and characterization of a new

family of cell-penetrating peptides: Cyclic cell-penetrating peptides. J Biol Chem.

2011;286:36932-36943. doi:10.1074/jbc.M111.264424

45. Contreras J, Elnagar AYO, Hamm-Alvarez SF, and Camarero JA. Cellular uptake of

cyclotide MCoTI-I follows multiple endocytic pathways. J Control Release.

2011;155:134-143. doi:10.1016/j.jconrel.2011.08.030

46. Henriques ST, Huang YH, Chaousis S, et al. The prototypic cyclotide Kalata B1 has a

unique mechanism of entering cells. Chem Biol. 2015;22:1087-2097.

doi:10.1016/j.chembiol.2015.07.012

*A paper describing the mechanism of cellular penetration of the cyclotide kalata

B1.

47. D’Souza C, Henriques ST, Wang CK, and Craik DJ. Structural parameters modulating

the cellular uptake of disulfide-rich cyclic cell-penetrating peptides: MCoTI-II and SFTI-

1. Euro J Med Chem. 2014;88:10-18. doi:10.1016/j.ejmech.2014.06.047

48. Nguyen GKT, Wang S, Qiu Y, et al. Butelase 1 is an Asx-specific ligase enabling peptide

macrocyclization and synthesis. Nat Chem Biol. 2014;10:732-738.

doi:10.1038/nchembio.1586

49. Harris KS, Durek T, Kaas Q, et al. Efficient backbone cyclization of linear peptides by a

recombinant asparaginyl endopeptidase. Nat Commun. 2015;6.

doi:10.1038/ncomms10199

50. Tang YQ, Yuan J, Osapay G, et al. A cyclic antimicrobial peptide produced in primate

leukocytes by the ligation of two truncated alpha-defensins. Science. 1999;286:498-502.

doi:10.1126/science.286.5439.498

51. Conibear AC, Rosengren KJ, Harvey PJ, and Craik DJ. Structural characterization of the

cyclic cystine ladder motif of θ-defensins. Biochemistry. 2012;51:9718-97126.

doi:10.1021/bi301363a

52. Conibear AC, Rosengren KJ, Daly NL, et al. The cyclic cystine ladder in theta-defensins

is important for structure and stability, but not antibacterial activity. J Biol Chem.

2013;288:10830-10840. doi:10.1074/jbc.M113.451047

53. Conibear AC and Craik DJ. The chemistry and biology of theta defensins. Angew Chem

Int Ed Engl. 2014;53:10612-10623. doi:10.1002/anie.201402167

54. Tongaonkar P, Tran P, Roberts K, et al. Rhesus macaque theta-defensin isoforms:

Expression, antimicrobial activities, and demonstration of a prominent role in neutrophil

granule microbicidal activities. J Leukoc Biol. 2011;89:283-290.

doi:10.1189/jlb.0910535

55. Schaal JB, Tran D, Tran P, et al. Rhesus macaque theta defensins suppress inflammatory

cytokines and enhance survival in mouse models of bacteremic sepsis. PLoS One.

2012;7:e51337. doi:10.1371/journal.pone.0051337

56. Wilmes M, Stockem M, Bierbaum G, et al. Killing of staphylococci by θ-defensins

involves membrane impairment and activation of autolytic enzymes. Antibiotics.

2014;3:617-631. doi:10.3390/antibiotics3040617

57. Tongaonkar P, Trinh KK, Schaal JB, et al. Rhesus macaque theta-defensin RTD-1

inhibits proinflammatory cytokine secretion and gene expression by inhibiting the

activation of NF-kappaB and MAPK pathways. J Leukoc Biol. 2015;98:1061-1070.

doi:10.1189/jlb.3A0315-102R

58. Wohlford-Lenane CL, Meyerholz DK, Perlman S, et al. Rhesus theta-defensin prevents

death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary

disease. J Virol. 2009;83:11385-11390. doi:10.1128/jvi.01363-09

59. Oh YT, Tran D, Buchanan TA, et al. theta-Defensin RTD-1 improves insulin action and

normalizes plasma glucose and FFA levels in diet-induced obese rats. Am J Physiol

Endocrinol Metab. 2015;309:e154-160. doi:10.1152/ajpendo.00131.2015

60. Cole AM, Hong T, Boo LM, et al. Retrocyclin: A primate peptide that protects cells from

infection by T- and M-tropic strains of HIV-1. Proc Natl Acad Sci USA. 2002;99:1813-

1818. doi:10.1073/pnas.052706399

61. Yang C, Boone L, Nguyen TX, et al. Theta-defensin pseudogenes in HIV-1-exposed,

persistently seronegative female sex-workers from Thailand. Infect Genet Evol.

2005;5:11-15. doi:10.1016/j.meegid.2004.05.006

62. Conibear AC, Bochen A, Rosengren KJ, et al. The cyclic cystine ladder of theta-

defensins as a stable, bifunctional scaffold: A proof-of-concept study using the integrin-

binding RGD motif. ChemBioChem. 2014;15:451-459. doi:10.1002/cbic.201300568

*The first paper demonstrating the use of θ-defensins as a stable scaffold for the

grafting of bioactive epitopes.

63. Aboye TL, Ha H, Majumder S, et al. Design of a Novel Cyclotide-Based CXCR4

Antagonist with Anti-Human Immunodeficiency Virus (HIV)-1 Activity. J Med Chem.

2012;55:10729-10734. doi:10.1021/jm301468k

64. Conibear AC, Chaousis S, Durek T, et al. Approaches to the stabilization of bioactive

epitopes by grafting and peptide cyclization. Biopolymers. 2016;106:89-100.

doi:10.1002/bip.22767

65. Luckett S, Garcia RS, Barker JJ, et al. High-resolution structure of a potent, cyclic

proteinase inhibitor from sunflower seeds. J Mol Biol. 1999;290:525-533.

doi:10.1006/jmbi.1999.2891

66. Elliott AG, Delay C, Liu H, et al. Evolutionary origins of a bioactive peptide buried

within preproalbumin. Plant Cell. 2014;26:981-995. doi:10.1105/tpc.114.123620

67. Qi RF, Song ZW, and Chi CW. Structural features and molecular evolution of bowman-

birk protease inhibitors and their potential application. Acta Biochim Biophys Sinica.

2005;37:283-292. doi:10.1111/j.1745-7270.2005.00048.x

68. Korsinczky ML, Clark RJ, and Craik DJ. Disulfide bond mutagenesis and the structure

and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1. Biochemistry.

2005;44:1145-1153. doi:10.1021/bi048297r

69. Craik DJ, Swedberg JE, Mylne JS, and Cemazar M. Cyclotides as a basis for drug design.

Exp Op Drug Disc. 2012;7:179-194. doi:10.1517/17460441.2012.661554

70. Chan LY, Craik DJ, and Daly NL. Cyclic thrombospondin-1 mimetics: Grafting of a

thrombospondin sequence into circular disulfide-rich frameworks to inhibit endothelial

cell migration. Biosci Rep. 2015;35. doi:10.1042/bsr20150210

71. Wang CK, Northfield SE, Huang YH, et al. Inhibition of tau aggregation using a

naturally-occurring cyclic peptide scaffold. Euro J Med Chem. 2016;109:342-349.

doi:10.1016/j.ejmech.2016.01.006

72. de Veer SJ, Swedberg JE, Akcan M, et al. Engineered protease inhibitors based on

sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and

conformation in Laskowski mechanism inhibition. Biochem J. 2015;469:243-253.

doi:10.1042/bj20150412

73. Fittler H, Depp A, Avrutina O, et al. Engineering a constrained peptidic scaffold towards

potent and selective furin inhibitors. ChemBioChem. 2015;16:2441-2444.

doi:10.1002/cbic.201500447

74. Jendrny C and Beck-Sickinger AG. Inhibition of kallikrein-related peptidases 7 and 5 by

grafting serpin reactive-center loop sequences onto sunflower trypsin inhibitor-1 (SFTI-

1). ChemBioChem. 2015;17:719-726. doi:10.1002/cbic.201500539

75. Swedberg JE, Nigon LV, Reid JC, et al. Substrate-guided design of a potent and selective

kallikrein-related peptidase inhibitor for kallikrein 4. Chem Biol 2009;16:633-643.

doi:10.1016/j.chembiol.2009.05.008

76. Okinyo-Owiti DP, Dong Q, Ling B, et al. Evaluating the cytotoxicity of flaxseed

orbitides for potential cancer treatment. Toxicol Rep. 2015;2:1014-1018.

doi:10.1016/j.toxrep.2015.06.011

77. Gaymes TJ, Cebrat M, Siemion IZ, and Kay JE. Cyclolinopeptide A (CLA) mediates its

immunosuppressive activity through cyclophilin-dependent calcineurin inactivation.

FEBS Letters. 1997;418:224-227. doi:10.1016/S0014-5793(97)01345-8

78. Katarzyńska J, Mazur A, Rudzińska E, et al. Cyclolinopeptide derivatives modify

methotrexate-induced suppression of the humoral immune response in mice. Euro J Med

Chem. 2011;46:4608-4617. doi:10.1016/j.ejmech.2011.07.040

79. Scott CP, Abel-Santos E, Wall M, et al. Production of cyclic peptides and proteins in

vivo. Proc Natl Acad Sci USA. 1999;96:13638-13643. doi:10.1073/pnas.96.24.13638

80. Smith JM, Frost JR, and Fasan R. Emerging strategies to access peptide macrocycles

from genetically encoded polypeptides. J Org Chem. 2013;78:3525-3531.

doi:10.1021/jo400119s

81. Passioura T, Katoh T, Goto Y, and Suga H. Selection-based discovery of druglike

macrocyclic peptides. Annu Rev Biochem. 2014;83:727-752. doi:doi:10.1146/annurev-

biochem-060713-035456

82. Lennard KR and Tavassoli A. Peptides come round: Using SICLOPPS libraries for early

stage drug discovery. Chemistry. 2014;20:10608-10614. doi:10.1002/chem.201403117

83. Clark RJ, Fischer H, Dempster L, et al. Engineering stable peptide toxins by means of

backbone cyclization: Stabilization of the alpha-conotoxin MII. Proc Natl Acad Sci USA.

2005;102:13767-13772. doi:10.1073/pnas.0504613102

84. Clark RJ, Jensen J, Nevin ST, et al. The engineering of an orally active conotoxin for the

treatment of neuropathic pain. Angew Chem Int Ed. 2010;49:6545-6548.

doi:10.1002/anie.201000620

85. Halai R, Callaghan B, Daly NL, et al. Effects of cyclization on stability, structure, and

activity of α-conotoxin RgIA at the α9α10 nicotinic acetylcholine receptor and GABAB

receptor. J Med Chem. 2011;54:6984-6992. doi:10.1021/jm201060r

86. Armishaw CJ, Dutton JL, Craik DJ, and Alewood PF. Establishing regiocontrol of

disulfide bond isomers of alpha-conotoxin ImI via the synthesis of N-to-C cyclic analogs.

Biopolymers. 2010;94:307-313. doi:10.1002/bip.21360

87. Armishaw CJ, Jensen AA, Balle LD, et al. Improving the stability of alpha-conotoxin

AuIB through N-to-C cyclization: The effect of linker length on stability and activity at

nicotinic acetylcholine receptors. Antioxid Redox Signal. 2011;14:65-76.

doi:10.1089/ars.2010.3458

88. Lovelace ES, Gunasekera S, Alvarmo C, et al. Stabilization of alpha-conotoxin AuIB:

Influences of disulfide connectivity and backbone cyclization. Antioxid Redox Signal.

2011;14:87-95. doi:10.1089/ars.2009.3068

89. Lovelace ES, Armishaw CJ, Colgrave ML, et al. Cyclic MrIA: A stable and potent cyclic

conotoxin with a aovel topological fold that targets the norepinephrine transporter. J Med

Chem. 2006;49:6561-6568. doi:10.1021/jm060299h

90. Hemu X, Taichi M, Qiu Y, et al. Biomimetic synthesis of cyclic peptides using novel

thioester surrogates. Biopolymers. 2013;100:492-501. doi:10.1002/bip.22308

91. Akcan M, Clark RJ, Daly NL, et al. Transforming conotoxins into cyclotides: Backbone

cyclization of P-superfamily conotoxins. Biopolymers. 2015;104:682-692.

doi:10.1002/bip.22699

92. Chan LY, Zhang VM, Huang YH, et al. Cyclization of the antimicrobial peptide gomesin

with native chemical ligation: Influences on stability and bioactivity. ChemBioChem.

2013;14:617-624. doi:10.1002/cbic.201300034

93. Jensen JE, Mobli M, Brust A, et al. Cyclisation increases the stability of the sea anemone

peptide APETx2 but decreases its activity at acid-sensing ion channel 3. Marine Drugs.

2012;10:1511-1527. doi:10.3390/md10071511

94. Clark RJ, Preza GC, Tan CC, et al. Design, synthesis, and characterization of cyclic

analogues of the iron regulatory peptide hormone hepcidin. Biopolymers. 2013;100:519-

526. doi:10.1002/bip.22350

95. Scanlon MJ, Naranjo D, Thomas L, et al. Solution structure and proposed binding

mechanism of a novel potassium channel toxin κ-conotoxin PVIIA. Structure.

1997;5:1585-1597. doi:10.1016/S0969-2126(97)00307-9

96. Kwon S, Bosmans F, Kaas Q, et al. Efficient enzymatic cyclization of an inhibitory

cystine knot-containing peptide. Biotechnol Bioeng. 2016;113:2202-2212.

doi:10.1002/bit.25993

97. Timmerman P, Beld J, Puijk WC, and Meloen RH. Rapid and quantitative cyclization of

multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces.

ChemBioChem. 2005;6:821-824. doi:10.1002/cbic.200400374

98. Chen S, Morales-Sanfrutos J, Angelini A, et al. Structurally diverse cyclisation linkers

impose different backbone conformations in bicyclic peptides. ChemBioChem.

2012;13:1032-1038. doi:10.1002/cbic.201200049

99. Chen S, Bertoldo D, Angelini A, et al. Peptide ligands stabilized by small molecules.

Angew Chem Int Ed Engl. 2014;53:1602-1606. doi:10.1002/anie.201309459

**An article highlighting the use of small organic molecules to stabilize peptide

epitopes through non-covalent bonding interactions.

100. Heinis C and Winter G. Encoded libraries of chemically modified peptides. Curr Opin

Chem Biol. 2015;26:89-98. doi:10.1016/j.cbpa.2015.02.008

**A review highlighting the development of peptides with topological constraints

using DNA-encoded protein libraries.

101. Heinis C, Rutherford T, Freund S, and Winter G. Phage-encoded combinatorial chemical

libraries based on bicyclic peptides. Nat Chem Biol. 2009;5:502-507.

doi:10.1038/nchembio.184

102. Baeriswyl V, Rapley H, Pollaro L, et al. Bicyclic peptides with optimized ring size inhibit

human plasma kallikrein and its orthologues while sparing paralogous proteases.

ChemMedChem. 2012;7:1173-1176. doi:10.1002/cmdc.201200071

103. Baeriswyl V, Calzavarini S, Chen S, et al. A synthetic factor XIIa inhibitor blocks

selectively intrinsic coagulation initiation. ACS Chem Biol. 2015;10:1861-1870.

doi:10.1021/acschembio.5b00103

*An article outlining the use of a constrained bicyclic peptide to potently and

selectively inhibit coagulation factor XIIa.

104. Bertoldo D, Khan MMG, Dessen P, et al. Phage selection of peptide macrocycles against

β-catenin to interfere with Wnt signaling. ChemMedChem. 2016;11:834-839.

doi:10.1002/cmdc.201500557

*A paper demonstrating the use of constrained bicyclic peptides to target β-catenin

interfering with the Wnt signaling pathway involved in tumor formation.

105. Pollaro L, Raghunathan S, Morales-Sanfrutos J, et al. Bicyclic peptides conjugated to an

albumin-binding tag diffuse efficiently into solid tumors. Mol Cancer Ther. 2015;14:151-

161. doi:10.1158/1535-7163.MCT-14-0534

106. Wallbrecher R, Depré L, Verdurmen WPR, et al. Exploration of the design principles of a

cell-penetrating bicylic peptide scaffold. Bioconjugate Chem. 2014;25:955-964.

doi:10.1021/bc500107f

**An article highlighting the structural parameters for cell penetration of

constrained bicyclic peptides.

107. Schafmeister CE, Po J, and Verdine GL. An all-hydrocarbon cross-linking system for

enhancing the helicity and metabolic stability of peptides. J Am Chem Soc.

2000;122:5891-5892. doi:10.1021/ja000563a

*The first paper reporting the development of a stapled peptide.

108. Aileron therapeutics successfully completes first-ever stapled peptide clinical trial.

Business Wire. Cambridge, Mass: Aileron Therapeutics, 2013. Available at:

http://www.businesswire.com/news/home/20130507005467/en/Aileron-Therapeutics-

Successfully-Completes-First-Ever-Stapled-Peptide [Last accessed 12 July 2016]

109. Aileron therapeutics initiates phase 1 cancer study of ALRN-6924 in advanced

hematologic and solid malignancies with wild type p53. Business Wire. Cambridge,

Mass: Aileron Therapeutics, 2015. Available at:

http://www.businesswire.com/news/home/20150212005199/en/Aileron-Therapeutics-

Initiates-Phase-1-Cancer-Study [Last accessed 12 July 2016]

110. Okamoto T, Zobel K, Fedorova A, et al. Stabilizing the pro-apoptotic BimBH3 helix

(BimSAHB) does not necessarily enhance affinity or biological activity. ACS Chem Biol.

2013;8:297-302. doi:10.1021/cb3005403

111. Li YC, Rodewald LW, Hoppmann C, et al. A versatile platform to analyze low-affinity

and transient protein-protein interactions in living cells in real time. Cell Rep.

2014;9:1946-1958. doi:10.1016/j.celrep.2014.10.058

*A paper describing a method to analyze weak intracellular protein-protein

interactions in real time and its application assessing the cellular permeability of

selected stapled peptides.

112. Cromm PM, Spiegel J, and Grossmann TN. Hydrocarbon stapled peptides as modulators

of biological function. ACS Chem Biol. 2015;10:1362-1375. doi:10.1021/cb501020r

*A review highlighting the development and applications of hydrocarbon stapled

peptides in drug discovery.

113. Chu Q, Moellering RE, Hilinski GJ, et al. Towards understanding cell penetration by

stapled peptides. MedChemComm. 2015;6:111-119. doi:10.1039/C4MD00131A

*A paper investigating the structural features associated with cell-penetrating

properties of stapled peptides.

114. Leshchiner ES, Braun CR, Bird GH, and Walensky LD. Direct activation of full-length

proapoptotic BAK. Proc Natl Acad Sci USA. 2013;110:e986–995.

doi:10.1073/pnas.1214313110

115. Bird GH, Gavathiotis E, LaBelle JL, et al. Distinct BimBH3 (BimSAHB) stapled

peptides for structural and cellular studies. ACS Chem Biol. 2014;9:831-837.

doi:10.1021/cb4003305

**An article highlighting the key design considerations in the development of

stapled peptides.

116. Bernal F, Wade M, Godes M, et al. A stapled p53 helix overcomes HDMX-mediated

suppression of p53. Cancer Cell. 2010;18:411-422. doi:10.1016/j.ccr.2010.10.024

*A study using phage display in the development of a stapled peptide that selectively

inhibits Mdm2/Mdm4 in the activation of the p53 tumor suppression protein.

117. Brown CJ, Quah ST, Jong J, et al. Stapled peptides with improved potency and

specificity that activate p53. ACS Chem Biol. 2013;8:506-512. doi:10.1021/cb3005148

118. Chang YS, Graves B, Guerlavais V, et al. Stapled α-helical peptide drug development: A

potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc Natl

Acad Sci USA. 2013;110:e3445-3454. doi:10.1073/pnas.1303002110

**A paper reporting the use of a stapled peptide that targets the overexpression of

inhibitory proteins MDM2 and MDMX to reactivate the p53 tumor suppressor

protein in vivo.

119. Long YQ, Huang SX, Zawahir Z, et al. Design of cell-permeable stapled peptides as

HIV-1 integrase inhibitors. J Med Chem. 2013;56:5601-5612. doi:10.1021/jm4006516

120. Phillips C, Roberts LR, Schade M, et al. Design and structure of stapled peptides binding

to estrogen receptors. J Am Chem Soc. 2011;133:9696-9699. doi:10.1021/ja202946k

121. Bird GH, Mazzola E, Opoku-Nsiah K, et al. Biophysical determinants for cellular uptake

of hydrocarbon-stapled peptide helices. Nat Chem Biol. 2016.

doi:10.1038/nchembio.2153

**A paper outlining the key design principles in the development of cell-permeable

stapled peptides.

122. Pelay-Gimeno M, Glas A, Koch O, and Grossmann TN. Structure-based design of

inhibitors of protein–protein interactions: Mimicking peptide binding epitopes. Angew

Chem Int Ed. 2015;54:8896-8927. doi:10.1002/anie.201412070

*A review that outlines current approaches in the development of various classes of

peptide mimetics targeting protein-protein interactions.

123. Obrecht D, Chevalier E, Moehle K, and Robinson JA. β-Hairpin protein epitope mimetic

technology in drug discovery. Drug Discov Today Technol. 2012;9:e63-69.

doi:10.1016/j.ddtec.2011.07.006

124. Späth J, Stuart F, Jiang L, and Robinson JA. Stabilization of a β-hairpin conformation in

a cyclic peptide using the templating effect of a heterochiral diproline unit. Helvetica

Chimica Acta. 1998;81:1726-1738. doi:10.1002/(SICI)1522-

2675(19980909)81:9<1726::AID-HLCA1726>3.0.CO;2-H

125. Schmidt J, Patora-Komisarska K, Moehle K, et al. Structural studies of β-hairpin

peptidomimetic antibiotics that target LptD in Pseudomonas sp. Bioorg Med Chem.

2013;21:5806-5810. doi:10.1016/j.bmc.2013.07.013

126. Srinivas N, Jetter P, Ueberbacher BJ, et al. Peptidomimetic antibiotics target outer-

membrane biogenesis in Pseudomonas aeruginosa. Science. 2010;327:1010-1013.

doi:10.1126/science.1182749

*A paper reporting the development of a β-hairpin peptidomimetic as an

antimicrobial agent against the pathogen Pseudomonas spp. with a novel mode of

action.

127. Vetterli SU, Moehle K, and Robinson JA. Synthesis and antimicrobial activity against

Pseudomonas aeruginosa of macrocyclic β-hairpin peptidomimetic antibiotics containing

N-methylated amino acids. Bioorg Med Chem. 2016. doi:10.1016/j.bmc.2016.05.027

128. Urfer M, Bogdanovic J, Lo Monte F, et al. A peptidomimetic antibiotic targets outer

membrane proteins and disrupts selectively the outer membrane in Escherichia coli. J

Biol Chem. 2016;291:1921-1932. doi:10.1074/jbc.M115.691725

**An article describing the development of a β-hairpin peptidomimetic that

selectively targets outer membrane β-barrel proteins in E. coli.

129. Fasan R, Dias RL, Moehle K, et al. Using a beta-hairpin to mimic an alpha-helix: cyclic

peptidomimetic inhibitors of the p53-HDM2 protein-protein interaction. Angew Chem Int

Ed Engl. 2004;43:2109-2112. doi:10.1002/anie.200353242

130. Fasan R, Dias RL, Moehle K, et al. Structure-activity studies in a family of beta-hairpin

protein epitope mimetic inhibitors of the p53-HDM2 protein-protein interaction.

ChemBioChem. 2006;7:515-526. doi:10.1002/cbic.200500452

131. Hill TA, Lohman R-J, Hoang HN, et al. Cyclic penta- and hexaleucine peptides without

N-methylation are orally absorbed. ACS Med Chem Lett. 2014;5:1148-1151.

doi:10.1021/ml5002823

132. Nielsen DS, Hoang HN, Lohman R-J, et al. Improving on nature: Making a cyclic

heptapeptide orally bioavailable. Angew Chem Int Ed. 2014;53:12059-12063.

doi:10.1002/anie.201405364

**A study that investigates the effects of heterocyclic constraints, intermolecular

hydrogen bonds and side-chain interactions in the development of orally

bioavailable cyclic peptides.

133. White TR, Renzelman CM, Rand AC, et al. On-resin N-methylation of cyclic peptides

for discovery of orally bioavailable scaffolds. Nat Chem Biol. 2011;7:810-817.

doi:10.1038/nchembio.664

*A paper describing a method for on-resin N-methylation and its application for the

development of orally bioavailable N-methylated cyclic peptides.

134. Beck JG, Chatterjee J, Laufer B, et al. Intestinal permeability of cyclic peptides:

Common key backbone motifs identified. J Am Chem Soc. 2012;134:12125-12133.

doi:10.1021/ja303200d

*Along with reference 135, highlights the backbone structural characteristics of

orally bioavailable cyclic peptides containing both N-methylated and D-amino acid

residues.

135. Marelli UK, Ovadia O, Frank AO, et al. cis-Peptide bonds: A key for intestinal

permeability of peptides? . Chem Euro J. 2015;21:15148-15152.

doi:10.1002/chem.201501600

136. Nielsen DS, Lohman R-J, Hoang HN, et al. Flexibility versus rigidity for orally

bioavailable cyclic hexapeptides. ChemBioChem. 2015;16:2289-2293.

doi:10.1002/cbic.201500441

137. Bockus AT, Lexa KW, Pye CR, et al. Probing the physicochemical boundaries of cell

permeability and oral bioavailability in lipophilic macrocycles inspired by natural

products. J Med Chem. 2015;58:4581-4589. doi:10.1021/acs.jmedchem.5b00128

138. Bockus AT, Schwochert JA, Pye CR, et al. Going out on a limb: Delineating the effects

of beta-branching, N-methylation, and side chain size on the passive permeability,

solubility, and flexibility of sanguinamide A analogues. J Med Chem. 2015;58:7409-

7418. doi:10.1021/acs.jmedchem.5b00919

Figure Captions

Figure 1. Schematic representation of the strategies used for the development of peptide

macrocycles in drug design. Bioactive elements can be utilized in drug design with a tailored

technology to develop novel leads that have enhanced pharmacological properties. This is

exemplified by the incorporation of bioactive epitopes with a macrocyclic scaffold (left) or the

stabilization of a α-helix with an organic link (center) or the cyclization of a bioactive peptide

using a peptide linker (right).

Figure 2. Schematic representation of the structural complexity of selected naturally occurring

cyclic peptides. From left to right: The orbitide segetalin B consisting of five residues and no

disulfides; the PawS-derived peptide sunflower trypsin inhibitor (SFTI-1) with 14 residues and a

single disulfide bond; the θ-defensin RTD-1 consisting of 18 residues and three parallel disulfide

bonds; the cyclotide kalata B1 consisting of 29 residues and three disulfide bonds in a cystine knot

arrangement.

Figure 3. Schematic representation of the cyclic cystine knot arrangement of the three cyclotide

subfamilies. The Möbius and bracelet subfamilies differ in the conformation of loop 5, whereas

the trypsin inhibitor family has substantial sequence and conformational differences from the other two subfamilies.

Figure 4. Schematic representation of the peptide macrocyclization strategies used to stabilize

peptides. (A) Representation of head-to-tail cyclization of the native conotoxin Vc1.1 through the

incorporation of a small linker sequence inserted between the N and C termini. (B) Schematic

representation of the stabilization strategy using a small organic linker to stabilize a bioactive

epitope. The three representations illustrate the use of CLIPS (chemical ligation of peptides onto

backbone) technology to make mono, bi and tri cyclic peptides stabilizing a bioactive

pharmacophore.

Figure 5. Schematic representation of two different stabilized secondary structures. (A) α,α-

Disubstituted non-natural amino acid building blocks used in the formation of stapled peptides

and the α-helical stapled peptides adjoined through hydrocarbon stapling of residues at position i to i+3, i to i+4, and i to i+7. (B) Generic structure of a β-hairpin mimetic stabilized by a template inducing a β-turn (D-Pro-L-Pro; dibenzofuran derivative; D-Pro-Gly).