Abstract

The use of disulfide-rich backbone-cyclized polypeptides, as molecular scaffolds to design a new generation of bioimaging tools and drugs that are potent and specific, and thus might have fewer side effects than traditional small-molecule drugs, is gaining increasing interest among the scientific and in the pharmaceutical industries. Highly constrained macrocyclic polypeptides are exceptionally more stable to chemical, thermal and biological degradation and show better biological activity when compared with their linear counterparts. Many of these relatively new scaffolds have been also found to be highly tolerant to sequence variability, aside from the conserved residues forming the disulfide bonds, able to cross cellular membranes and modulate intracellular protein–protein interactions both in vitro and in vivo. These properties make them ideal tools for many biotechnological applications. The present study provides an overview of the new developments on the use of several disulfide-rich backbone-cyclized polypeptides, including cyclotides, θ-defensins and sunflower trypsin inhibitor peptides, in the development of novel bioimaging reagents and therapeutic leads.

Introduction

The genomic and proteomic revolutions have provided us with an ever-increasing number of mechanistic insights into human diseases [14]. Mutated genes and pathologic protein products are emerging as the basis for the development of novel therapeutic agents to treat human diseases such as cancer or autoimmune diseases. However, the selective disruption of protein–protein interactions (PPIs) still remains a very challenging task, as the interacting surfaces are relatively large and relatively flat [57]. In fact, among the most intractable of targets are those involving intracellular PPIs, which require the therapeutic agent to efficiently cross the cell membrane [8,9].

Broadly speaking, there are only two major structural classes of approved drugs: small molecules and protein therapeutics (also known as biologics). Small molecules typically show good stability and good pharmacological properties, but their intrinsic small size (≤100 atoms) endows them with only a modest overall surface area available to contact a protein target. Accordingly, the identification of small molecules able to efficiently disrupt PPIs presents significant challenges [10,11]. Protein-based therapeutics, however, possess high specificity/selectivity and high affinity for protein targets [12]. The use of therapeutic monoclonal antibodies to target extracellular protein receptors is just one example [13,14]. Antibodies, however, suffer from clear limitations: they are expensive to produce, cannot be delivered orally, show low tissue penetration and are unable to reach intracellular targets [15]. The potential problems associated with the use of antibody fragments have led to the exploration of alternative protein scaffolds as a source for novel protein-based therapeutics [1623]. However, the utility of protein-based therapeutics has been typically hampered by their generally poor stability and limited bioavailability [24]. To overcome these limitations, special attention has been recently given to the use of highly constrained peptides, also known as micro-proteins, for the modulation of PPIs [2527].

In this review, we will present the recent developments in the use of Cys-rich backbone-cyclized polypeptides for targeting PPIs. Circular Cys-rich peptides are widely distributed natural product polypeptides among different species including animals and plants (Scheme 1). They have attractive features including thermal, chemical and biological stability against proteases. Peptides in this group include cyclotides, mammalian θ-defensins and sunflower protease inhibitors.

Biotechnological applications of disulfide-rich backbone cyclized polypeptides. Schematic representation 2026.

Scheme 1.
Biotechnological applications of disulfide-rich backbone cyclized polypeptides. Schematic representation 2026.

Schematic representation of the structural complexity of the naturally occurring disulfide-rich backbone-cyclized peptides that are commonly used as tools for the development of novel therapeutic leads (top right, depicting a cyclotide able to activate the p53 pathway in cancer cells in vivo) [9] and novel bioimaging agents (bottom right, depicting a cyclotide able to visualize CXCR4-expressing tumors in vivo) [28] and molecular structures shown on the left include cyclotides (cycloviolacin O2, pdb: 1NBJ) [29], θ-defensins (RTD-1, pdb: 1HVZ) [30] and sunflower peptide trypsin inhibitors (SFTI-1, pdb: 1JBL) [31].

Scheme 1.
Biotechnological applications of disulfide-rich backbone cyclized polypeptides. Schematic representation 2026.

Schematic representation of the structural complexity of the naturally occurring disulfide-rich backbone-cyclized peptides that are commonly used as tools for the development of novel therapeutic leads (top right, depicting a cyclotide able to activate the p53 pathway in cancer cells in vivo) [9] and novel bioimaging agents (bottom right, depicting a cyclotide able to visualize CXCR4-expressing tumors in vivo) [28] and molecular structures shown on the left include cyclotides (cycloviolacin O2, pdb: 1NBJ) [29], θ-defensins (RTD-1, pdb: 1HVZ) [30] and sunflower peptide trypsin inhibitors (SFTI-1, pdb: 1JBL) [31].

Cyclotides

Cyclotides are fascinating micro-proteins (≈30 residues long) present in plants from the Violaceae, Rubiaceae, Cucurbitaceae, and more recently Fabaceae and Solanaceae families [32]. They display various biological properties such as protease inhibitory, antimicrobial, insecticidal, cytotoxic, anti-HIV and hormone-like activities (see refs [25,26] for recent reviews of the biotechnological applications of cyclotides). They share a unique head-to-tail cyclic cystine-knot (CCK) topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine-knot topology (Figure 1) [29]. Cyclotides can be considered as natural combinatorial peptide libraries structurally constrained by the cystine-knot scaffold and head-to-tail cyclization but in which hypermutation of essentially all residues is permitted with the exception of the strictly conserved cysteines that comprise the knot [3335]. The main features of cyclotides are a remarkable stability due to the cystine knot, a small size making them readily accessible to chemical synthesis and an excellent tolerance to sequence variations. For example, the first cyclotide to be discovered, kalata B1, is an orally effective uterotonic [36], and other cyclotides have been also shown to be orally bioavailable [37,38] and capable of crossing cell membranes [39,40] to efficiently target extracellular [28,41,42] and intracellular PPIs in vivo [9]. In addition, cyclotides have been shown to be poorly immunogenic due to their highly constrained nature [18,43]. Cyclotides thus appear as highly promising leads or frameworks for peptide drug design [26,4446].

Sequence alignment and structures of different cyclotides belonging to the Möbius (kalata B1, pdb: 1NB1) [29], bracelet (cycloviolacin O1, pdb: 1NBJ) [29] and trypsin inhibitor (MCoTI-II, pdb: 1IB9) [47] subfamilies.

Figure 1.
Sequence alignment and structures of different cyclotides belonging to the Möbius (kalata B1, pdb: 1NB1) [29], bracelet (cycloviolacin O1, pdb: 1NBJ) [29] and trypsin inhibitor (MCoTI-II, pdb: 1IB9) [47] subfamilies.

Disulfide connectivities and backbone-cyclization are shown in red and orange, respectively. The six Cys residues are labeled with roman numerals, whereas loops connecting the different Cys residues are designated with arabic numerals. Conserved Cys and Asp/Asn (required for backbone-cyclization in nature) residues are marked in yellow and light blue, respectively. Molecular graphics were created using Yasara (www.yasara.org).

Figure 1.
Sequence alignment and structures of different cyclotides belonging to the Möbius (kalata B1, pdb: 1NB1) [29], bracelet (cycloviolacin O1, pdb: 1NBJ) [29] and trypsin inhibitor (MCoTI-II, pdb: 1IB9) [47] subfamilies.

Disulfide connectivities and backbone-cyclization are shown in red and orange, respectively. The six Cys residues are labeled with roman numerals, whereas loops connecting the different Cys residues are designated with arabic numerals. Conserved Cys and Asp/Asn (required for backbone-cyclization in nature) residues are marked in yellow and light blue, respectively. Molecular graphics were created using Yasara (www.yasara.org).

Cyclotides are classified into three subfamilies known as the Möbius, bracelet and trypsin inhibitor cyclotide subfamilies [32]. All the subfamilies have the same CCK topology, although the composition of the loops is slightly different (Figure 1). Bracelet cyclotides are usually larger and more structurally diverse than Möbius cyclotides, and they make-up ∼60% of all the cyclotides known so far [48]. Bracelet cyclotides are more difficult to obtain by chemical synthesis than either Möbius or trypsin inhibitor cyclotides due to the difficulties associated with fold them correctly in vitro [49]. Owing to this, bracelet cyclotides have been less used as molecular scaffolds to target PPIs.

Cyclotides from the trypsin inhibitor subfamily are found in the seeds from several plants of Momordica genus [50,51] and as their name indicates potent trypsin inhibitors [52]. This is the cyclotide subfamily with fewer members identified thus far showing little sequence homology with the other cyclotides beyond the presence of the CCK fold. In fact, these cyclotides seem related to linear cystine-knot squash trypsin inhibitors and sometimes are also referred to as cyclic knottins [53]. Cyclotides from this family possess a longer sequence in loop 1, making the Cys-knot slightly less rigid than in cyclotides from the other two subfamilies.

Naturally occurring cyclotides are produced in plants from dedicated genes that can encode multiple copies of the same cyclotide or even mixtures of different cyclotide sequences [54]. Recent studies indicate that an asparaginyl endopeptidase (AEP)-like ligase is responsible for the backbone-cyclization of cyclotides [5557]; however, the complete mechanism of how the precursor cyclotide protein is processed at its N-terminus has not completely elucidated yet. Cyclotides can also be chemically synthesized using solid-phase peptide synthesis and native chemical ligation as they are relatively small polypeptides containing typically ∼30–40 residues (see [58] for a recent review on the chemical synthesis of cyclotides). More recently, the use of protein splicing in cis and trans has also allowed the production of fully folded cyclotides inside bacterial and yeast cells using heterologous expression systems [59,60].

Table 1 summarizes recent examples on the use of the cyclotide scaffold to target PPIs. The first two examples demonstrated that the pharmacological potential of engineered cyclotides was aimed for the development of novel peptide-based novel anticancer [61,62] and anti-viral peptide-based therapeutics [63]. The development of molecules targeting angiogenesis has been shown to potential therapeutic avenues in cancer treatment as tumor growth is usually associated with unregulated angiogenesis. The molecular grafting of an Arg-rich peptide antagonist of the vascular endothelial growth factor A (VEGF-A) receptor onto several loops of the Möbius cyclotide kalata B1 yielded antagonists with low µM activity for blocking VEGF activity [61]. This was one of the first examples where a bioactive peptide was used to produce a novel cyclotide with a specific biological activity; however, it should be noted that the biological activity would still need to be improved by several orders of magnitude for a potential pharmacological application in vivo. A similar approach using the molecular framework of kalata B1 was recently used in the development of cyclotides able to modulate the bradykinin and melanocortin 4 receptors for pain and obesity management, respectively [37,64]. It is worth mentioning that one of the designed kalata B1-based bradykinin antagonists in this work was shown to be orally bioavailable [37]. In this context, a recent study using a point mutated kalata B1 cyclotide was also reported to have oral bioavailability and in a mouse model of multiple sclerosis [38]. This finding highlights the potential of the cyclotide molecular framework for the development of novel orally bioavailable peptide-based therapeutics.

Table 1
Summary of work published in engineered cyclotides with novel biological activities leading to therapeutic and bioimaging applications1
Cyclotide Biological activity Loop modified Application Ref. 
Möbius subfamily 
 Kalata B1 VEGF-A antagonist 2, 3, 5 and 6 Antiangiogenic, potential anti-cancer activity [61
 Kalata B1 Dengue NS2B–NS3 protease inhibitor 2 and 5 Anti-viral for Dengue virus infections [65
 Kalata B1 Bradikynin B1 receptor antagonist Chronic and inflammatory pain [37
 Kalata B1 Melanocortin 4 receptor Agonist Obesity [64
 Kalata B1 Neuropilin-1/2 antagonist 5 and 6 Inhibition of endothelial cell migration and angiogenesis [66
 Kalata B1 Immunomodulator 5 and 6 Protecting against multiple sclerosis [67
 Kalata B1 Immunomodulator Protecting against multiple sclerosis [68
Trypsin inhibitor subfamily 
 MCoTI-I CXCR4 antagonist Anti-metastatic and anti-HIV PET–CT imaging [28,41,42
 MCoTI-I p53-Hdm2/HdmX antagonist Anti-tumor by activation of p53 pathway [9
 MCoTI-II FMDV 3C protease Inhibitor Anti-viral for foot-and-mouth disease [63
 MCoTI-II β-Tryptase inhibitor 3, 5 & 6 Inflammation diseases [69
 MCoTI-II β-Tryptase inhibitor
Human elastase inhibitor 
Inflammation diseases [62
 MCoTI-II CTLA-4 antagonist 1,3 and 6 Immunotherapy for cancer [70
 MCoTI-II Tryptase inhibitor Anti-cancer [71
 MCoTI-II VEGF receptor agonist Wound healing and cardiovascular damage [72
 MCoTI-I α-Synuclein-induced cytotoxicity inhibitor Parkinson's disease
Validate phenotypic screening of genetically encoded cyclotide libraries 
[60
 MCoTI-II BCR-Abl kinase inhibitor 1 and 6 Chronic myeloid leukemia
Attempt to graft both a cell-penetrating peptide and a kinase inhibitor 
[73
 MCoTI-I MAS1 receptor agonist Lung cancer and myocardial infarction [74
 MCoTI-II SET antagonist Potential anticancer [75
 MCoTI-II FXIIa and FXa inhibitors 1 and 6 Antithrombotic and cardiovascular disease [76
 MCoTI-II Thrombospondin-1 (TSP-1) agonist Microvascular endothelial cell migration inhibition
Anti-angiogenesis 
[77
 MCoTI-II Antiangiogenic 5 and 6 Anti-cancer [78
Cyclotide Biological activity Loop modified Application Ref. 
Möbius subfamily 
 Kalata B1 VEGF-A antagonist 2, 3, 5 and 6 Antiangiogenic, potential anti-cancer activity [61
 Kalata B1 Dengue NS2B–NS3 protease inhibitor 2 and 5 Anti-viral for Dengue virus infections [65
 Kalata B1 Bradikynin B1 receptor antagonist Chronic and inflammatory pain [37
 Kalata B1 Melanocortin 4 receptor Agonist Obesity [64
 Kalata B1 Neuropilin-1/2 antagonist 5 and 6 Inhibition of endothelial cell migration and angiogenesis [66
 Kalata B1 Immunomodulator 5 and 6 Protecting against multiple sclerosis [67
 Kalata B1 Immunomodulator Protecting against multiple sclerosis [68
Trypsin inhibitor subfamily 
 MCoTI-I CXCR4 antagonist Anti-metastatic and anti-HIV PET–CT imaging [28,41,42
 MCoTI-I p53-Hdm2/HdmX antagonist Anti-tumor by activation of p53 pathway [9
 MCoTI-II FMDV 3C protease Inhibitor Anti-viral for foot-and-mouth disease [63
 MCoTI-II β-Tryptase inhibitor 3, 5 & 6 Inflammation diseases [69
 MCoTI-II β-Tryptase inhibitor
Human elastase inhibitor 
Inflammation diseases [62
 MCoTI-II CTLA-4 antagonist 1,3 and 6 Immunotherapy for cancer [70
 MCoTI-II Tryptase inhibitor Anti-cancer [71
 MCoTI-II VEGF receptor agonist Wound healing and cardiovascular damage [72
 MCoTI-I α-Synuclein-induced cytotoxicity inhibitor Parkinson's disease
Validate phenotypic screening of genetically encoded cyclotide libraries 
[60
 MCoTI-II BCR-Abl kinase inhibitor 1 and 6 Chronic myeloid leukemia
Attempt to graft both a cell-penetrating peptide and a kinase inhibitor 
[73
 MCoTI-I MAS1 receptor agonist Lung cancer and myocardial infarction [74
 MCoTI-II SET antagonist Potential anticancer [75
 MCoTI-II FXIIa and FXa inhibitors 1 and 6 Antithrombotic and cardiovascular disease [76
 MCoTI-II Thrombospondin-1 (TSP-1) agonist Microvascular endothelial cell migration inhibition
Anti-angiogenesis 
[77
 MCoTI-II Antiangiogenic 5 and 6 Anti-cancer [78
1

Adapted from references [26,79].

The cyclotides from the trypsin inhibitory subfamily have also used as templates to engineer cyclotides with novel biological activities by means of molecular grafting. For example, the cyclotide MCoTI-I has been recently used for the design of a potent CXCR4 antagonist [41]. Overexpression of the CXCR4 receptor has been observed in multiple cancers where it is believed that it promotes metastasis, angiogenesis, and tumor growth and/or survival [80].

Cyclotides from the trypsin inhibitor subfamily have been also used for the development of protease inhibitors with pharmacological relevance. The cyclotide MCoTI-II was transformed into a potent and selective foot-and-mouth disease (FMDV) 3C protease inhibitor by introducing mutations onto loops 1 and 6 [63]. A similar approach was used by the same authors to generate β-tryptase and human leukocyte elastase inhibitors with low nM Ki values [62,69]. These results suggest that MCoTI-cyclotides provide a versatile molecular framework for the development of protease inhibitors against targets in viral and inflammatory diseases.

The most exciting feature of the cyclotide scaffold is that some members of the trypsin inhibitor subfamily can cross the cellular membrane of mammalian cells, therefore, making possible the intracellular delivery of biologically active cyclotides to target intracellular PPIs [39,40]. Our group was recently able to generate a potent activator (low nM) of p53 function by inhibiting the interaction between p53 and the proteins Hdm2/HdmX using the cyclotide MCoTI-I as molecular scaffold (Figure 2) [9]. The resulting cyclotide MCo-PMI was able to bind with high affinity the p53-binding domains of both Hdm2 and HdmX, showed high ex vivo stability in serum and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo (Figure 2) [9]. This work represents the first example showing an engineered cyclotide able to target an intracellular PPI in an animal model of prostate cancer, therefore, highlighting the therapeutic potential of MCoTI-cyclotides for targeting intracellular PPIs. Exactly the same approach but using cyclotide MCoTI-II instead has been also employed to obtain an antagonist for the SET protein that is overexpressed in some human cancers [75]. The cyclotide MCoTI-II has been also used as a molecular framework to design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL. This was accomplished by grafting sequences derived from an optimal substrate for the Abl kinase onto cyclotide MCoTI-II [73]. Several grafted cyclotides were able to show Abl kinase inhibition in vitro in the low micromolar range; however, they did not show significant growth inhibition in a human chronic myeloid leukemia (CML) cell line. Although the inhibition of cytosolic Abl was not investigated in this work, the lack of cellular growth inhibition could suggest that more potent kinase inhibitors may need to be designed to observe activity in cell-based assays and/or animal models. In our experience, when designing bioactive MCoTI-based cyclotides, it should be desirable to achieve in vitro activities in the low to mid-nM range to obtain bioactive cyclotides in cell-based assays and/or animal models [9].

Structure and in vivo activity of the first cyclotide designed to antagonize an intracellular protein–protein interaction in vivo [9].

Figure 2.
Structure and in vivo activity of the first cyclotide designed to antagonize an intracellular protein–protein interaction in vivo [9].

(A) Solution structure of the engineered cyclotide MCo-PMI (magenta) and its intracellular molecular target, the p53-binding domain of oncogene Hdm2 (blue) (pdb: 2M86) [9]. The cyclotide binds with low nM affinity to both the p53-binding domains of Hdm2 and HdmX. (B) Cyclotide MCo-PMI activates the p53 tumor suppressor pathway and blocks tumor growth in a human colorectal carcinoma xenograft mouse model. (C) Tumor samples were subjected to SDS–PAGE and analyzed by western blotting for p53, Hdm2 and p21, indicating the activation of p53 on tumor tissue.

Figure 2.
Structure and in vivo activity of the first cyclotide designed to antagonize an intracellular protein–protein interaction in vivo [9].

(A) Solution structure of the engineered cyclotide MCo-PMI (magenta) and its intracellular molecular target, the p53-binding domain of oncogene Hdm2 (blue) (pdb: 2M86) [9]. The cyclotide binds with low nM affinity to both the p53-binding domains of Hdm2 and HdmX. (B) Cyclotide MCo-PMI activates the p53 tumor suppressor pathway and blocks tumor growth in a human colorectal carcinoma xenograft mouse model. (C) Tumor samples were subjected to SDS–PAGE and analyzed by western blotting for p53, Hdm2 and p21, indicating the activation of p53 on tumor tissue.

Our group has also recently shown that cyclotides can be obtained by heterologous expression using both prokaryotic and eukaryotic expression systems [59,60]. For example, a novel MCoTI-grafted cyclotide (MCoCP4) was able to inhibit α-synuclein-induced cytotoxicity in yeast Saccharomyces cerevisiae [60]. α-Synuclein is a small lipid-binding protein that is prone to misfolding and aggregation and has been linked to Parkinson's disease making it a validated therapeutic target for Parkinson's disease.

The ability to produce natively folded cyclotides in the cell [60,81,82], as described earlier, makes possible the generation and rapid screening of large libraries of cyclotides, potentially containing billions of members, that are genetically encoded cyclotides. The generation of such tremendous molecular diversity permits the development of selection of strategies that mimic the evolutionary processes found in nature for the selection of novel cyclotide sequences able to target specific molecular targets. For example, as proof of principle, our group used in cell expression of a small library based in cyclotide MCoTI-II in Escherichia coli where every residue in loops 1, 2, 3, 4 and 5 was mutated to explore the effects on folding and trypsin-binding activity of the resulting mutants [82]. This early study revealed that most of the mutations did not affect folding on the resulting cyclotides, therefore, emphasizing the high plasticity and sequence tolerance of MCoTI-based cyclotides [82].

The use of an acyclic version of cyclotide kalata B1 was employed for the screening and selection of novel cyclotides specific for the VEGF-A-binding site on neuropilin-1 [66]. This study used bacterial display libraries and the authors were able to obtain kalata-based cyclotides with high affinity (Kd ≈ 50 nM), increased protease resistance and conferred improved potency for inhibiting endothelial cell migration in vitro (EC50 ≈ 100 nM) [66]. A yeast surface display approach of an acyclic version of cyclotide MCoTI-II was also employed for the screening of a linearized cyclotide library to select strong binders cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma [70].

More recently, a cyclotide-based library was employed for phenotypic screening in eukaryotic cells [60]. In this work, an engineered cyclotide (MCoCP4) that was designed to reduce the toxicity of human α-synuclein in live yeast cells was selected by phenotypic screening from cells transformed with a mixture of plasmids encoding MCoCP4 and inactive cyclotide MCoTI-I in a ratio of 1 : 50 000. These results show the potential for performing rapid phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells for the selection of bioactive compounds. These exciting results demonstrate the potential to perform phenotypic screening of genetically encoded cyclotide-based libraries in eukaryotic cells for the rapid selection of novel bioactive cyclotides. Moreover, expression in eukaryotic systems should allow the production of cyclotides with different post-translational modifications not available in bacterial expression systems.

The development of efficient methods for the chemical synthesis, cyclization and folding of cyclotides has also made possible to perform high-throughput screening on chemically generated libraries of cyclotides [42]. A small library of MCoTI-based CXCR4 cyclotide antagonists was chemically obtained using a ‘tea-bag’ approach in combination with high efficiency folding protocols [42]. The approach described in this work also included an efficient purification procedure to rapidly remove non-folded or partially folded cyclotides from the cyclization-folding crude. This approach can be also employed for the purification of cyclotide mixtures, thereby allowing the synthesis of amino acid and positional scanning libraries to perform efficient screening of large chemical-generated libraries [83].

The potential of bioactive cyclotide to be employed as bioimaging agents has also been recently explored and reported for the first time [28]. An MCoTI-based CXCR4 antagonist cyclotide (MCo-CVX-6D) was shown to be an excellent bioimaging tool to visualize CXCR4-overexpressing cancer cells in a mouse model. A [64Cu]-DOTA-labeled version of cyclotide MCo-CVX-6D was used for the efficient detection of tumors containing CXCR4-expressing cells in mice using positron emission tomography–computed tomography (PET–CT) (Figure 3) [28]. These results demonstrate the in vivo specificity and retention of a bioactive molecularly targeted cyclotide and highlight the potential of bioactive cyclotides for the development of new imaging agents that target CXCR4 [28].

Use of a CXCR4-targeting cyclotide as a bioimaging tool for detecting CXCR4-overexpressing tumor cells in animal models [28].

Figure 3.
Use of a CXCR4-targeting cyclotide as a bioimaging tool for detecting CXCR4-overexpressing tumor cells in animal models [28].

(A) Distribution of [64Cu]MCo-CVX-6D (CXCR4-targetign cyclotide) and [64Cu]MCoTI-ID (a DOTA-labeled variant of native trypsin inhibitor cyclotide MCoTI-I) in NOD/SCID mice bearing U87 and U87-stb-CXCR4 tumors with PET–CT. (B) Ex vivo evaluation of [64Cu]MCo-CVX-6D and [64Cu]MCoTI-ID distribution and specificity in NOD/SCID mice bearing U87 and U87-stb-CXCR4 tumors. Ex vivo biodistribution analysis was performed at 90 min and 24 h after post-tracers injection.

Figure 3.
Use of a CXCR4-targeting cyclotide as a bioimaging tool for detecting CXCR4-overexpressing tumor cells in animal models [28].

(A) Distribution of [64Cu]MCo-CVX-6D (CXCR4-targetign cyclotide) and [64Cu]MCoTI-ID (a DOTA-labeled variant of native trypsin inhibitor cyclotide MCoTI-I) in NOD/SCID mice bearing U87 and U87-stb-CXCR4 tumors with PET–CT. (B) Ex vivo evaluation of [64Cu]MCo-CVX-6D and [64Cu]MCoTI-ID distribution and specificity in NOD/SCID mice bearing U87 and U87-stb-CXCR4 tumors. Ex vivo biodistribution analysis was performed at 90 min and 24 h after post-tracers injection.

Given the good pharmacological properties of some bioactive cyclotides, their biodistribution has been recently studied [28,84]. These studies indicate that MCoTI-cyclotides are distributed predominantly to the serum and kidneys, confirming that they are stable in serum and suggesting that they are eliminated from the blood through renal clearance (Figure 3B) [28,84]. However, no significant uptake into the brain was observed for cyclotide MCoTI-II [84], which suggests that MCoTI-cyclotides may require the engineering of this scaffold by either grafting and/or conjugating appropriate molecular motifs enabling its transport through the blood–brain barrier. This would open the possibility for the development of cyclotides able to target molecular interactions in the central nervous system.

Mammalian θ-defensins

Defensins are cysteine-rich antimicrobial peptides (AMPs) that play a critical role in the innate immune defense of mammals [8587]. Although they are classically known for their antimicrobial activities, they have been also shown to be involved in other defense mechanisms including immune modulation, neutralization of endotoxins and anticancer properties [87,88]. Mammalian defensins are peptides containing mostly β-sheet structures, six Cys residues forming three intramolecular disulfides and a high content on positively charged residues. They have been classified into α-, β- and θ-defensins depending on their overall structure [89,90]. In contrast with α- and β-defensins, θ-defensins are backbone-cyclized peptides formed by the head-to-tail covalent assembly of two nonapeptides derived from α-defensin-related precursors (Figure 4) [85,91]. θ-Defensins are, to date, the only known cyclic polypeptides expressed in animals [85].

Sequence and structure of naturally occurring disulfide-rich backbone-cyclized peptide θ-defensin RTD-1 (pdb: 1HVZ) [30].

Figure 4.
Sequence and structure of naturally occurring disulfide-rich backbone-cyclized peptide θ-defensin RTD-1 (pdb: 1HVZ) [30].

The backbone-cyclized peptide (connecting bond shown in orange) is stabilized by the three disulfide bonds in a ladder formation (disulfide bonds shown in yellow). Molecular graphic was created using Yasara (www.yasara.org).

Figure 4.
Sequence and structure of naturally occurring disulfide-rich backbone-cyclized peptide θ-defensin RTD-1 (pdb: 1HVZ) [30].

The backbone-cyclized peptide (connecting bond shown in orange) is stabilized by the three disulfide bonds in a ladder formation (disulfide bonds shown in yellow). Molecular graphic was created using Yasara (www.yasara.org).

θ-Defensins have shown to possess antimicrobial activity against both Gram-positive and Gram-negative bacteria [85], as well as anti-fungal [85] and anti-HIV [92,93] activities.

Chemically produced human θ-defensin, derived from human pseudogene sequences, have also shown to protect human cells from infection by HIV-1 [92] and have been even evaluated as a topical anti-HIV agent [9496]. θ-Defensins have been shown to have moderate activity against several bacterial toxins and proteases [97], as well as human metalloproteases like TNF-α-converting enzyme (TACE) [83]. θ-Defensins have also been shown to possess anti-inflammatory properties in animal models [98]. In addition, θ-defensins present high resistance to proteolytic degradation in serum and plasma due to the network of disulfide bonds and backbone-cyclized topology [83,98]. Altogether, these unique features have made θ-defensin, an ideal molecular framework for the development of novel peptide-based therapeutics and bioimaging tools (Table 2) [98,99].

Table 2
Summary of work published in engineered θ-defensin RTD-1 and backbone-cyclized trypsin inhibitor SFTI-1 peptides with novel biological activities with potential therapeutic and bioimaging applications
Peptide Biological activity Application Ref. 
RTD-1 Lethal factor protease inhibitor
TNFα-converting protease inhibitor 
Anthrax toxin antidote
Biosensing
Anti-inflammatory 
[83]
[83,98
RTD-1 αvβ3 integrin agonist Antiangiogenic
Anticancer
Bioimaging 
[100
RTD-1 RTD-1-based genetically encoded libraries (complexity ≈ 2 × 107High-throughput screening of novel biological activities [101
SFTI-1 Mesotrypsin inhibitor Anti-cancer
Anti-metastatic 
[102
SFTI-1 KLK4 protease inhibitor Anti-cancer anti-metastatic [103105
SFT-1 KLK5 and KLK7 protease
inhibitors 
Anti-cancer
Skin diseases: psoriasis and atopic dermatitis 
[106
SFTI-1 αvβ6 integrin agonist Anti-cancer
Bioimaging 
[107,108
SFTI-1 Dll4 agonist Angiogenesis marker
Bioimaging 
[109
SFTI-1 Antiangiogenic Anti-cancer [78
SFTI-1 Furin protease
Inhibitor 
Autoimmune diseases
Inflammatory diseases
Anti-cancer 
[110
SFTI-1 Annexin A1 protein-like activity Inflammatory bowel diseases [111
SFTI-1 Melanocortin 1 receptor
Agonist 
Obesity and inflammatory disorders [112
SFTI-1 Scavenger for anticitrullinated protein/peptide autoantibodies Diagnostic and/or treatment of rheumatoid arthritis [113
SFTI-1 Bradykinin receptor antagonist Pain treatment [114
Peptide Biological activity Application Ref. 
RTD-1 Lethal factor protease inhibitor
TNFα-converting protease inhibitor 
Anthrax toxin antidote
Biosensing
Anti-inflammatory 
[83]
[83,98
RTD-1 αvβ3 integrin agonist Antiangiogenic
Anticancer
Bioimaging 
[100
RTD-1 RTD-1-based genetically encoded libraries (complexity ≈ 2 × 107High-throughput screening of novel biological activities [101
SFTI-1 Mesotrypsin inhibitor Anti-cancer
Anti-metastatic 
[102
SFTI-1 KLK4 protease inhibitor Anti-cancer anti-metastatic [103105
SFT-1 KLK5 and KLK7 protease
inhibitors 
Anti-cancer
Skin diseases: psoriasis and atopic dermatitis 
[106
SFTI-1 αvβ6 integrin agonist Anti-cancer
Bioimaging 
[107,108
SFTI-1 Dll4 agonist Angiogenesis marker
Bioimaging 
[109
SFTI-1 Antiangiogenic Anti-cancer [78
SFTI-1 Furin protease
Inhibitor 
Autoimmune diseases
Inflammatory diseases
Anti-cancer 
[110
SFTI-1 Annexin A1 protein-like activity Inflammatory bowel diseases [111
SFTI-1 Melanocortin 1 receptor
Agonist 
Obesity and inflammatory disorders [112
SFTI-1 Scavenger for anticitrullinated protein/peptide autoantibodies Diagnostic and/or treatment of rheumatoid arthritis [113
SFTI-1 Bradykinin receptor antagonist Pain treatment [114

Our group has developed efficient approaches for the chemical and recombinant production of θ-defensins [83,91,101,115,116]. Using a ‘tea-bag’ approach [117] in combination with a one-pot cyclization method involving native chemical ligation and oxidative folding, our group has obtained potent θ-defensins analogs able to inhibit anthrax lethal factor (LF) and TACE with Ki values ≈40 and ≈157 nM, respectively [83]. It is worth mentioning that these analogs showed also significant activity in the presence of 0.1% bovine serum albumin (BSA) [83]. The use of BSA at this concentration is widely used in high-throughput screening assays to avoid the selection on nonspecific inhibitors as well as to test activity in conditions mimicking those found in complex biological environments [83]. The θ-defensin scaffold has also recently employed for designing integrin antagonists [100]. This was accomplished by grafting the integrin-binding Arg-Gly-Asp (RGD) peptide motif into the θ-defensin RTD-1 molecular framework. The most active compound had an IC50 of ≈18 nM for the αvβ3 integrin and presented high serum stability.

More recently, natively folded and bioactive θ-defensin RTD-1 has been produced in high yield (0.7 mg of RTD-1 per gram of wet cells) inside E. coli cells by making use of intracellular protein trans-splicing in combination with a high efficient split-intein [101]. This approach was used to generate a genetically encoded RTD-1-based peptide library in live E. coli cells encoding ≈2 × 107 different RTD-1-based sequences. These results clearly demonstrate the possibility of using genetically encoded RTD-1-based peptide libraries in live E. coli cells, which is a critical first step for developing in-cell screening and directed evolution technologies using the cyclic peptide RTD-1 as a molecular scaffold for screening novel biological activities.

In summary, these results clearly show the high robustness of the θ-defensin scaffold for the generation of molecular diversity providing also a stable and conformationally restrained scaffold for bioactive epitopes in a β-strand or turn conformation.

Sunflower trypsin inhibitor 1

Sunflower trypsin inhibitor 1 (SFTI-1) is a 14 amino acid backbone-cyclized peptide containing a single disulfide bond that is naturally found in the seeds of sunflower (Helianthus annuus) [118]. SFTI-1 belongs to the Bowman-Birk inhibitor (BBI) family whose members are found in many plants and are potent serine protease inhibitors [119]. Structural analysis of SFTI-1 shows a well-defined double β-hairpin loop linked by two short antiparallel β-strands (Figure 5) [48,118,120]. The backbone-cyclized SFTI-1 is the smallest and the most potent protease member of the family with a Ki against trypsin in the low nM range [118,119].

Sequence and structure of naturally occurring disulfide-rich backbone-cyclized and SFTI-1 (pdb: 1JBL) [121].

Figure 5.
Sequence and structure of naturally occurring disulfide-rich backbone-cyclized and SFTI-1 (pdb: 1JBL) [121].

SFTI-1 contains one disulfide bond (shown in yellow) and a backbone-cyclized topology (connecting bond shown in orange). Molecular structure was created using Yasara (www.yasara.org).

Figure 5.
Sequence and structure of naturally occurring disulfide-rich backbone-cyclized and SFTI-1 (pdb: 1JBL) [121].

SFTI-1 contains one disulfide bond (shown in yellow) and a backbone-cyclized topology (connecting bond shown in orange). Molecular structure was created using Yasara (www.yasara.org).

The relatively rigid backbone of SFTI-1 makes its protease-binding loop well defined, which can serve as a general scaffold for serine protease inhibitors [122]. For example, introduction of several mutations within this loop has produced SFTI-based analogs able to inhibit a variety of serine proteases [71,103,106,123,124]. The structural features of SFTI-1 characterized by the presence of a backbone-cyclization combined with an internal disulfide bond and an extensive hydrogen-binding network make it exceptionally stable to thermal or enzymatic degradation [71,125]. SFTI-1 has been also shown to be nontoxic and be able to cross cellular membranes [39]. Moreover, as shown with other backbone-cyclized disulfide-rich scaffolds, the molecular framework provided by SFTI-1 can also be readily re-engineered by grafting foreign biological active peptide sequences into one of the loops producing SFTI-analogs with novel biological activities (Table 2) [72,77,109,126].

Native SFTI-1 has been found to be a potent inhibitor of matriptase (Ki ≈ 1 nM) [127]. Matriptase is a type II transmembrane serine protease found in most cancer cells, where it has been shown that can activate key pro-metastatic substrates to trigger cell migration, cancer invasion and metastasis, therefore providing a target of therapeutic intervention [128]. Amino acid scanning of the position P2′ residue (Ile) in native SFTI-1 has been also accomplished to produce a potent mesotrypsin inhibitor [102]. Replacement of this residue by aromatic residues (Tyr, Phe, Trp or the non-canonical amino acid 4,4′-biphenyl-l-alanine (Bip)) yielded an SFTI-I analog that maintained a similar structure to native SFTI-1 and showed marked improvements in activity against mesotrypsin (exceeding 100-fold) [102].

SFTI-1 has also been re-engineered to produce potent human kallikrein-related peptidase 4 (KLK4) inhibitor [103]. Human KLK4 is a potential target for prostate cancer treatment because of its proteolytic ability to activate many tumorigenic and metastatic pathways including the protease-activated receptors (PARs) [121,129]. In this work, SFTI-1 was modified by introducing several mutations (K5R, T4Q and R2F), identified by using a combination of molecular modeling and sparse matrix substrate screening, to produce the analog SFTI-FCQR that was potent and selective KLK4 inhibitor (Ki ≈ 3 nM) [103]. Further optimization of the KLK4 inhibitor SFTI-FCQR by the same authors using molecular dynamic algorithms produced a significantly improved inhibitor (SFTI-FCQR Asn14) with a Ki value of ≈64 pM [104]. The same authors have also employed a similar approach to improve the weaker activity of SFTI-I against cathepsin G (Ki ≈ 570 nM) through optimization of the binding loop to generate molecules with higher potency (Ki = 1.6 nM) and higher selectivity (over 360-fold) for other related proteases [105].

The development of SFTI-based inhibitors of kallikrein-related peptidases 7 (KLK7) and 5 (KLK5) was also recently accomplished by grafting of the reactive-center loop (RCL) of several serpins reported to inhibit KLK5 and KLK7 [106]. KLK7 and KLK5 are expressed in human skin, and their dysregulation is associated with skin diseases such as Netherton syndrome, atopic dermatitis and psoriasis [130]; and different types of human cancer [131]. The best SFTI-derived inhibitors against KLK7 and KLK5 provide Ki values ranging from 0.6 to 0.9 µM values.

The SFTI-1 scaffold has been recently used in combination with phage-display techniques to screen and select a novel SFTI-based ITG αv β6-binding peptide (SFITGv6) [107]. The ITG αvβ6 receptor is highly expressed on head and neck squamous cell carcinoma (HNSCC) and is the target of the antiangiogenic RGD-peptide cilengitide [132]. In this work, a linearized version of SFITGv6 peptide labeled with the radiotracer 177Lu-DOTA was employed as a bioimaging agent for ITG αv β6-positive carcinomas [107,108]. SFTI-1 has also been employed to graft the binding loop of micro-protein Min-23 selected to bind angiogenesis marker delta-like ligand4 (Dll4) by using a phage-display approach [109]. The micro-protein Min-23 is a two disulfide-bridge stabilized scaffold, which was rationally designed by miniaturization of its parent knottin Ecballium elaterium trypsin inhibitor II (EETI-II) [133]. In this work, the resulting grafter SFTI-derived peptide was able to preserve the Dll4-binding specificity and the tumor-targeting capability of the original micro-protein [109]. A similar grafting approach using antiangiogenic peptides was also able to yield potent antiangiogenic SFTI-derived peptides [78]. The grafted peptide SFTI-PEDF displayed an almost similar degree of activity as the linear grafted peptide with a better proteolytic stability profile. SFTI-1 has also been transformed into a furin inhibitor by replacing the trypsin-binding loop of SFTI-1 by a natural furin substrate [110]. The resulting grafted SFTI-based peptide was further optimized by using computer-based structural modeling to generate a sub-nanomolar furin inhibitor (Ki ≈ 0.5 nM) that showed very good selectivity over trypsin (>10 000) and matriptase (>1000) [110].

The SFTI-1 molecular framework was recently employed to produce a cyclic peptide for reducing inflammation in models of inflammatory bowel diseases (IBDs) [111]. In this work, the authors grafted a small bioactive peptide from the annexin A1 protein into trypsin-binding loop of SFTI-1. The resulting SFTI-based peptide (cyc-MC12) maintained the overall fold of the naturally occurring cyclic peptide as more effective at reducing inflammation in a mouse model of acute colitis than the bioactive peptide alone showing enhanced ex vivo stability in human serum.

The SFTI-1 scaffold was also recently employed for the production of a novel subtype of selective melanocortin receptor (MCR) agonists [112]. This was accomplished by grafting the α/β/γ-melanocyte-stimulating hormone (MSH)-derived HFRW tetrapeptide into the different loops of SFTI-1 in combination with systematic N-methylation of the grafted pharmacophore. One of the double N-methylated SFTI-derived peptides was able to show low nM activity for human MC1R being ∼100 times more selective for this receptor than for MC3R. The nuclear magnetic resonance structural analysis of this grafted peptide revealed the key role of peptide bond N-methylation in shaping the conformation of the grafted peptide pharmacophore. This work highlights the potential of cyclic peptide scaffolds for epitope grafting in combination with N-methylation to introduce receptor subtype selectivity in the context of peptide-based drug discovery [112].

An interesting application of bioactive grafted SFTI-based peptides was recently reported as a scavenger for anticitrullinated protein/peptide autoantibodies (ACPA) for potential diagnostic and/or treatment of rheumatoid arthritis [113]. In this interesting work, the SFTI-1 scaffold was engineered to display a citrulline-containing ACPA-binding epitope identified from the α-chain of human fibrinogen. The resulting cyclic peptide scavenger showed high apparent affinity and subtype-specific binding for ACPA, as well as superior serum stability.

Native SFTI-1 has been recently used to produce the orally active and metabolically stable analgesic cyclic peptide, which was created by grafting the analgesic bradykinin receptor antagonist peptide into the trypsin inhibitory loop of SFTI-1 [114]. The resulting SFTI-based peptide, called TIBA, was protected from degradation by exopeptidases as well as the endopeptidases, showing an ex vivo serum half-life of more than 6 h. The SFTI-grafted peptide TIBA was also found to be orally active in an animal pain model using a hot plate assay, therefore providing a promising lead for the design of a novel type of peptide-based analgesics.

All these examples show the great potential of SFTI-1 as a molecular framework for grafting or engineering new activities. Its small size and high resistance to proteolytic degradation are making it increasingly recognized as an excellent template for engineering studies as it does not require structural optimization and the inhibitor's inherent activity can be re-directed to other serine proteases by substituting residues that form major binding contacts.

Concluding remarks

It is fair to say that the use of highly constrained disulfide-rich backbone-cyclized polypeptides is becoming to gain acceptance as molecular scaffolds for the potential design of novel peptide-based therapeutics and diagnostic tools [25,26,44]. This is, in part, to their unique properties which include their small size that allows chemical synthesis, the ability to be expressed using standard expression systems [58,90,134], high resistance to chemical, physical and biological degradation and, in some cases, the ability to cross cellular membranes to target intracellular and extracellular PPIs [9,37,38].

Of the three types of scaffolds reviewed here, the cyclotide scaffold without question is one of the more exciting. The cyclotide unique knotted arrangement of three disulfide bonds and exceptional tolerance sequence variation in all their hypervariable loops provides a unique molecular platform to design novel cyclotides with new biological activities by rational design using molecular grafting techniques or by employing molecular evolution techniques making use of the multiple loops found on the cyclotide scaffold [26,44,79]. This unique feature should allow using multiple loops in a synergistic fashion to design cyclotides able to target with higher affinity and selectivity specific biomolecular interactions. Cyclotides have also been shown to cross mammalian cellular membranes to target PPIs in vitro, and also more importantly in animal models [9]. This highlights the high stability of the Cys-knot to be degraded/oxidized under complex biological conditions.

As mentioned earlier, the relative small size of these scaffolds facilitates their chemical synthesis allowing the introduction of chemical modifications such as non-natural amino acids, such as N-methylated [112] and d-amino acids [28,41,74], and potential PEGylation to improve their pharmacological properties. None of the scaffolds reviewed here have reached human clinical trials yet; however, the results obtained with several bioactive compounds in animal models may hint that this could occur in a not too distant future. One of the main challenges that affect this type of constrained polypeptides is their oral bioavailability if they want to be competitive with small-molecule therapeutics. Although we have seen that some cyclotides [3638] and SFTI-based [114] peptides have been shown to be orally active, there is still little information about their oral bioavailability. It is anticipated, however, that more studies on the biopharmaceutical properties of these exciting new peptide-based molecular scaffolds will be available very soon.

Abbreviations

     
  • ACPA

    anticitrullinated protein/peptide autoantibodies

  •  
  • BSA

    bovine serum albumin

  •  
  • CCK

    cyclic cystine knot

  •  
  • CTLA-4

    cytotoxic T lymphocyte-associated antigen 4

  •  
  • Dll4

    delta-like ligand4

  •  
  • FMDV

    foot-and-mouth disease

  •  
  • KLK4

    kallikrein-related peptidase 4

  •  
  • KLK5

    kallikrein-related peptidase 5

  •  
  • KLK7

    kallikrein-related peptidase 7

  •  
  • PET–CT

    positron emission tomography–computed tomography

  •  
  • PPIs

    protein–protein interactions

  •  
  • RGD

    Arg-Gly-Asp

  •  
  • SFITGv6

    SFTI-based ITG αv β6-binding peptide

  •  
  • SFTI-1

    Sunflower trypsin inhibitor 1

  •  
  • TACE

    TNF-α-converting enzyme

  •  
  • VEGF-A

    vascular endothelial growth factor A

Funding

This work was supported by National Institutes of Health Research Grant [R01-GM113363], Department of Defense Congressionally Directed Medical Research Programs in Lung Cancer Grant [LC150051], BROAD Medical Research Program-Crohn's and Colitis Foundation of America [grant #483566], Lupus Research Institute and Whittier Foundation.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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