Immunosuppressive Cyclotides: A Promising Approach for Treating Autoimmune Diseases.

The immune system maintains constant surveillance to prevent the infiltration of both endogenous and exogenous threats into host organisms. The process is regulated by effector immune cells that combat external pathogens and regulatory immune cells that inhibit excessive internal body inflammation, ultimately establishing a state of homeostasis within the body. Disruption to this process could lead to autoimmunity, which is often associated with the malfunction of both T cells and B cells with T cells playing a more major role. A number of therapeutic mediators for autoimmune diseases are available, from conventional disease-modifying drugs to biologic agents and small molecule inhibitors. Recently, ribosomally synthesized peptides, specifically cyclotides from plants are currently attracting more attention as potential autoimmune disease therapeutics due to their decreased toxicity compared to small molecules inhibitors as well as their remarkable stability against a number of factors. This review provides a concise overview of various cyclotides exhibiting immunomodulatory properties and their potential as therapeutic interventions for autoimmune diseases.

Insights into the synthesis strategies of plant-derived cyclotides.

Cyclotides are plant peptides characterized with a head-to-tail cyclized backbone and three interlocking disulfide bonds, known as a cyclic cysteine knot. Despite the variations in cyclotides peptide sequences, this core structure is conserved, underlying their most useful feature: stability against thermal and chemical breakdown. Cyclotides are the only natural peptides known to date that are orally bioavailable and able to cross cell membranes. Cyclotides also display bioactivities that have been exploited and expanded to develop as potential therapeutic reagents for a wide range of conditions (e.g., HIV, inflammatory conditions, multiple sclerosis, etc.). As such, in vitro production of cyclotides is of the utmost importance since it could assist further research on this peptide class, specifically the structure-activity relationship and its mechanism of action. The information obtained could be utilized to assist drug development and optimization. Here, we discuss several strategies for the synthesis of cyclotides using both chemical and biological routes.

Two dimensional materials are non-nanotoxic and biocompatible towards cyclotides: evidence from classical molecular dynamics simulations.

Cyclotides are backbone-cyclized peptides of plant origin enriched with disulfide bonds, having exceptional stability towards thermal denaturation and proteolytic degradation. They have a plethora of activities like antibacterial, antifungal, anti-tumor and anti-HIV properties predominantly owing to their selective interaction with certain phospholipids, thereby leading to the disruption of cellular membranes. On the other hand, low-dimensional materials like graphene and hexagonal boron nitride (h-BN) are also known to show membrane-proliferating activities through lipid extraction. A plausible and more effective antibacterial, anti-tumor and antifungal agent would be a composite of these 2D materials and cyclotides, provided the structures of the peptides remain unperturbed upon adsorption and interaction. In this study, classical molecular dynamics simulations are performed to understand the nature of adsorption of cyclotides belonging to different families on graphene and h-BN and analyze the resulting structural changes. It is revealed that, due to their exceptional structural stability, cyclotides maintain their structural integrity upon adsorption on the 2D materials. In addition, the aggregated states of the cyclotides, which are ubiquitous in plant organs, are also not disrupted upon adsorption. Extensive free energy calculations show that the adsorption strength of the cyclotides is moderate in comparison to those of other similar-sized biomolecules, and the larger the size of the aggregates, the weaker the binding of individual peptides with the 2D materials, thereby leading to their lower release times from the materials. It is predicted that graphene and h-BN may safely be used for the preparation of composites with cyclotides, which in turn may be envisaged to be probable candidates for manufacturing next-generation bionano agents for agricultural, antibacterial and therapeutic applications.

An insight into biological activities of native cyclotides for potential applications in agriculture and pharmaceutics.

Cyclotides are plant-derived mini-proteins of 28 - 37 amino acids. They have a characteristic head-to-tail cyclic backbone and three disulfide cross-linkages formed by six highly conserved cysteine residues, creating a unique knotted ring structure, known as a cyclic cystine knot (CCK) motif. The CCK topology confers immense stability to cyclotides with resistance to thermal and enzymatic degradation. Native cyclotides are of interest due to their multiple biological activities with several potential applications in agricultural (e.g. biopesticides, antifungal) and pharmaceutical (e.g. anti-HIV, cytotoxic to tumor cells) sectors. The most recent application of insecticidal activity of cyclotides is the commercially available biopesticidal spray known as 'Sero X' for cotton crops. Cyclotides have a general mode of action and their potency of bioactivity is determined through their binding ability, pore formation and disruption of the target biological membranes. Keeping in view the important potential applications of biological activities of cyclotides and the lack of an extensive and analytical compilation of bioactive cyclotides, the present review systematically describes eight major biological activities of the native cyclotides from four angiosperm families viz. Fabaceae, Poaceae, Rubiaceae, Violaceae. The bioactivities of 94 cytotoxic, 57 antibacterial, 44 hemolytic, 25 antifungal, 21 anti-HIV, 20 nematocidal, 10 insecticidal and 5 molluscicidal cyclotides have been comprehensively elaborated. Further, their distribution in angiosperm families, mode of action and future prospects have also been discussed.

Natural and grafted cyclotides in cancer therapy: An insight.

Cyclotides is a rapidly growing class of plant-derived cyclic peptides exhibiting several bioactivities with potential applications in the agricultural and pharmaceutical sectors. Both natural and grafted cyclotides have shown promise in cancer therapy. Approximately 70 natural cyclotides belonging to three plant families (Fabaceae, Rubiaceae, and Violaceae) have shown cytotoxicity against several cancer cell lines. Cyclotides exhibit considerable stability against thermal and enzymatic proteolysis, owing to their unique structure with knotted topology and head to tail cyclization. Further, their small size, high stability, oral bioavailability, and tolerance to amino acid substitution in structural loops make them an ideal platform for designing peptide-based drugs for cancer. Thus, cyclotides provide ideal scaffolds for bioactive epitope grafting and facilitating drug delivery in cancer treatment. Many anticancer linear peptides have been grafted in cysteine knotted cyclic framework of cyclotide for enhancing their cell permeability across cellular membranes, thereby improving their delivery and pharmacokinetics. The present review comprehensively discusses the distribution, toxicity, and anticancer bioactivity of natural cyclotides. Further, it systematically elaborates on the role and action of epitopes' into grafted cyclotides in targeting cancer. The review also encompasses related patents landscape study and future challenges in peptide-based cancer therapy.

Strategies to Enhance Metabolic Stabilities.

Macrocyclic peptides are a unique class of molecules that display a relatively constrained peptidic backbone as compared to their linear counterparts leading to the defined 3-D orientation of the constituent amino acids (pharmacophore). Although they are attractive candidates for lead discovery owing to the unique conformational features, their peptidic backbone is susceptible to proteolytic cleavage in various biological fluids that compromise their efficacy. In this chapter we review the various classical and contemporary chemical and biological approaches that have been utilized to combat the metabolic instability of macrocyclic peptides. We note that any chemical modification that helps in providing either local or global conformational rigidity to these macrocyclic peptides aids in improving their metabolic stability typically by slowing the cleavage kinetics by the proteases.

Building upon Nature's Framework: Overview of Key Strategies Toward Increasing Drug-Like Properties of Natural Product Cyclopeptides and Macrocycles.

The pharmaceutical industry has focused mainly in the development of small-molecule entities intended for oral administration for the past decades. As a result, the majority of existing drugs address only a narrow range of biological targets. In the era of post-genomics, transcriptomics, and proteomics, there is an increasing interest on larger modulators of proteins that can span larger surfaces, access new therapeutic mechanisms of action, and provide greater target specificity. Traditional drug-like molecules developed using "rule-of-five" (Ro5) guidelines have been proven ineffective against a variety of challenging targets, such as protein-protein interactions, nucleic acid complexes, and antibacterial modalities. However, natural products are known to be effective at modulating such targets, leading to a renewed focus by medicinal chemists on investigating underrepresented chemical scaffolds associated with natural products. Here we describe recent efforts toward identification of novel natural cyclopeptides and macrocycles as well as selected medicinal chemistry strategies to increase drug-like properties or further exploration of their activity.

Using backbone-cyclized Cys-rich polypeptides as molecular scaffolds to target protein-protein interactions.

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.

Cyclotides: Overview and Biotechnological Applications.

Cyclotides are globular microproteins with a unique head-to-tail cyclized backbone, stabilized by three disulfide bonds forming a cystine knot. This unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to chemical, thermal, and biological degradation compared to other peptides of similar size. In addition, cyclotides have been shown to be highly tolerant to sequence variability, aside from the conserved residues forming the cystine knot. Cyclotides can also cross cellular membranes and are able to modulate intracellular protein-protein interactions, both in vitro and in vivo. All of these features make cyclotides highly promising as leads or frameworks for the design of peptide-based diagnostic and therapeutic tools. This article provides an overview on cyclotides and their applications as molecular imaging agents and peptide-based therapeutics.

Immunostimulating and Gram-negative-specific antibacterial cyclotides from the butterfly pea (Clitoria ternatea).

UNLABELLED: Cyclotides are plant-derived, cyclic miniproteins with three interlocking disulfide bonds that have attracted great interests because of their excellent stability and potential as peptide therapeutics. In this study, we characterize the cyclotides of the medicinal plant Clitoria ternatea (butterfly pea) and investigate their biological activities. Using a combined proteomic and transcriptomic method, we identified 41 novel cyclotide sequences, which we named cliotides, making C. ternatea one of the richest cyclotide-producing plants to date. Selected members of the cationic cliotides display potent antibacterial activity specifically against Gram-negative bacteria with minimal inhibitory concentrations as low as 0.5 µm. Remarkably, they also possess prominent immunostimulating activity. At a concentration of 1 µm, cationic cliotides are capable of augmenting the secretion of various cytokines and chemokines in human monocytes at both resting and lipopolysaccharide-stimulated states. Chemokines such as macrophage inflammatory proteins 1α and 1ß, interferon γ-induced protein 10, interleukin 8 and tumor necrosis factor α were among the most upregulated with up to 129-fold increase in secretion level. These findings suggest cyclotides can serve as potential candidates for novel immunomodulating therapeutics. DATABASE: The protein sequences reported in this paper (cT13-cT21) are available in the UniProt Knowledgebase under the accession numbers C0HJS0, C0HJS1, C0HJS2, C0HJS3, C0HJS4, C0HJS5, C0HJS6, C0HJS7 and C0HJS8, respectively. The transcriptome data in this paper are available at the Sequence Read Archive database (NCBI) under accession number SRR1613316. The protein precursors reported in this paper (ctc13, ctc15, ctc17-ctc19, ctc21-ctc53) are available at GenBank under the accession numbers KT732712, KT732713, KT732714, KT732715, KT732716, KT732717, KT732718, KT732719, KT732720, KT732721, KT732722, KT732723, KT732724, KT732725, KT732726, KT732727, KT732728, KT732729, KT732730, KT732731, KT732732, KT732733, KT732734, KT732735, KT732736, KT732737, KT732738, KT732739, KT732740, KT732741, KT732742, KT732743, KT732744, KT732745, KT732746, KT732747, KT732748 and KT732749, respectively.

Plant antimicrobial peptides as potential anticancer agents.

Antimicrobial peptides (AMPs) are part of the innate immune defense mechanism of many organisms and are promising candidates to treat infections caused by pathogenic bacteria to animals and humans. AMPs also display anticancer activities because of their ability to inactivate a wide range of cancer cells. Cancer remains a cause of high morbidity and mortality worldwide. Therefore, the development of methods for its control is desirable. Attractive alternatives include plant AMP thionins, defensins, and cyclotides, which have anticancer activities. Here, we provide an overview of plant AMPs anticancer activities, with an emphasis on their mode of action, their selectivity, and their efficacy.

Cystine-knot peptides: emerging tools for cancer imaging and therapy.

Cystine-knot miniproteins, also known as knottins, constitute a large family of structurally related peptides with diverse amino acid sequences and biological functions. Knottins have emerged as attractive candidates for drug development as they potentially fill a niche between small molecules and protein biologics, offering drug-like properties and the ability to bind to clinical targets with high affinity and selectivity. Due to their extremely high stability and unique structural features, knottins also demonstrate promise in addressing challenging drug development goals, including the potential for oral delivery and the ability to access intracellular drug targets. Several naturally-occurring knottins have recently received approval for treating chronic pain and irritable bowel syndrome, while others are under development for tumor imaging applications. To expand beyond nature's repertoire, rational and combinatorial protein engineering methods are generating tumor-targeting knottins for use as cancer diagnostics and therapeutics.

Immunosuppressive peptides and their therapeutic applications.

The immune system is vital for detecting and evading endogenous and exogenous threats to the body. Failure to regulate this homeostasis leads to autoimmunity, which is often associated with malfunctioning T cell signaling. Several medications are available to suppress over-reactive T lymphocytes, but many of the currently marketed drugs produce severe and life-threatening side-effects. Ribosomally synthesized peptides are gaining recognition from the pharmaceutical industry for their enhanced selectivity and decreased toxicity compared with small molecules; in particular, circular peptides exhibit remarkable stability and increased oral administration properties. For example, plant cyclotides effectively inhibit T lymphocyte proliferation. They are composed of a head-to-tail cyclized backbone and a cystine-knot motif, which confers them with remarkable stability, thus making them attractive pharmaceutical tools.

In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide.

The overexpression of Hdm2 and HdmX is a common mechanism used by many tumor cells to inactive the p53 tumor suppressor pathway promoting cell survival. Targeting Hdm2 and HdmX has emerged as a validated therapeutic strategy for treating cancers with wild-type p53. Small linear peptides mimicking the N-terminal fragment of p53 have been shown to be potent Hdm2/HdmX antagonists. The potential therapeutic use of these peptides, however, is limited by their poor stability and bioavailability. Here, we report the engineering of the cyclotide MCoTI-I to efficiently antagonize intracellular p53 degradation. The resulting cyclotide MCo-PMI was able to bind with low nanomolar affinity to both Hdm2 and HdmX, showed high stability in human serum, and was cytotoxic to wild-type p53 cancer cell lines by activating the p53 tumor suppressor pathway both in vitro and in vivo. These features make the cyclotide MCoTI-I an optimal scaffold for targeting intracellular protein-protein interactions.

Cyclotides: a patent review.

INTRODUCTION: Cyclotides are bioactive mini-proteins from plants that have the unique topological feature of a head-to-tail cyclic backbone combined with a cystine knot. Because of this structure they are ultra-stable and have attracted interest as peptide-based templates for drug design applications. Cyclotide biosynthesis involves processing from a genetically encoded precursor protein but methods have been developed for their man-made synthesis using solid phase peptide synthesis as well as recombinant methods. Their natural function in plants is as insecticidal agents and thus they have potential applications in agriculture. However, they have a range of pharmaceutically relevant activities, including anti-HIV, antimicrobial and uterotonic activity. Their exceptional stability and facile synthesis lend them to uses as pharmaceutical templates into which bioactive peptide sequences can be grafted. AREAS COVERED: This article reviews the patent literature associated with cyclotides with a focus on therapeutic applications. These patents are primarily related to the use of cystine knot scaffolds for the production of peptide-based drug leads, molecular probes or diagnostic agents. EXPERT OPINION: Although no cyclotide-related peptide has yet reached clinical trials, proof-of-concept has been obtained that bioactive peptide sequences can be grafted onto a cyclotide framework, maintaining biological activity while becoming resistant to proteolysis. Thus, cyclotides are promising templates in drug development applications and there is increasing interest in them and related cystine knot scaffolds, as well as in the use of other disulfide-rich scaffolds, in drug design.

Biological activities of natural and engineered cyclotides, a novel molecular scaffold for peptide-based therapeutics.

Cyclotides are a growing family of large plant-derived backbone-cyclized polypeptides (≈30 amino acids long) that share a disulfide-stabilized core characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds makes them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Currently more than 100 sequences of different cyclotides have been characterized and the number is expected to increase dramatically in the coming years. Considering their stability, biological activities and ability to cross the cell membrane, cyclotides can be exploited to develop new peptide-based drugs with high potential for success. The cyclotide scaffold can be engineered or evolved using molecular evolution to inhibit protein-protein interactions implicated in cancer and other human diseases, or design new antimicrobial. The present review reports the biological diversity and therapeutic potential of natural and engineered cyclotides.

Cyclotides, a promising molecular scaffold for peptide-based therapeutics.

Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (approximately 30 amino acids long) that share a disulfide-stabilized core (three disulfide bonds) characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds make them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Currently, more than 100 sequences of different cyclotides have been characterized, and the number is expected to increase dramatically in the coming years. Considering their stability and biological activities like anti-HIV, uterotonic, and insecticidal, and also their abilities to cross the cell membrane, cyclotides can be exploited to develop new stable peptide-based drugs. We have recently demonstrated the intriguing possibility of producing libraries of cyclotides inside living bacterial cells. This opens the possibility to generate large genetically encoded libraries of cyclotides that can then be screened inside the cell for selecting particular biological activities in a high-throughput fashion. The present minireview reports the efforts carried out toward the selection of cyclotide-based compounds with specific biological activities for drug design.

Cyclotides as templates in drug design.

Cyclotides are remarkably stable proteins from plants that have a range of pharmaceutical and agricultural applications based on both their various bioactivities and their potential for use as stable protein-engineering templates. This article discusses literature on pharmaceutically relevant activities of cyclotides, including anti-HIV, antimicrobial and cytotoxic activities, and evaluates their potential therapeutic applications. Their applications as templates for the design of antiangiogenic agents for the treatment of cancer and as anti-infective agents are also described. Toxic effects of cyclotides, whose native function is as insecticidal agents, can be removed by simple mutagenesis, thus rationalizing the apparent conundrum of proposing insecticidal agents as leads for human therapeutics.

Design and therapeutic applications of cyclotides.

Cyclotides are plant-derived peptides with a cyclic backbone and knotted topology of disulfide bonds. Their extreme stability and natural sequence variation has led to the suggestion that they might be useful as scaffolds to stabilize bioactive sequences. Recent studies have shown that anti-angiogenic activity and protease inhibitory activity against a foot and mouth disease protease can be grafted onto the cyclotide framework. There has also been significant progress made in determining the mechanism of cyclization of cyclotides and in producing cyclotides using bacterial expression and plant cell culture. There is a wide range of disease states that can be targeted using the cyclotide framework and the advances that have been made in the production of cyclotides will facilitate their development as pharmaceutical templates.