Boosting the anti-tumor activity of natural killer cells by caripe 8 - A Carapichea ipecacuanha isolated cyclotide.

Cyclotides are head-to-tail cyclized peptides with a unique cystine-knot motif. Their structure provides exceptional resistance against enzymatic, chemical, or thermal degradation compared to other peptides. Peptide-based therapeutics promise high specificity, selectivity and lower immunogenicity, making them safer alternatives to small molecules or large biologicals. Cyclotides were researched due to their anti-cancer properties by inducing apoptosis in tumor cells in the past, but the impact of cyclotides on cytotoxic immune cells was poorly studied. Natural Killer (NK) cells are cytotoxic innate lymphoid cells and play an important role in the defense against infected, stressed and transformed cells. NK cells do not need prior sensitization and act in an antigen independent manner, holding promising potential in the field of immunotherapy. To investigate the effect of immunomodulatory cyclotides on NK cells, we evaluated several peptide-enriched plant extracts on NK cell mediated cytotoxicity. We observed that the extract samples derived from Carapichea ipecacuanha (Brot.) L. Andersson augments the killing potential of mouse NK cells against different tumor targets in vitro. Subsequent isolation of cyclotides from C. ipecacuanha extracts led to the identification of a primary candidate that enhances cytotoxicity of both mouse and human NK cells. The augmented killing is facilitated by the increased degranulation capacity of NK cells. In addition, we noted a direct toxic effect of caripe 8 on tumor cells, suggesting a dual therapeutic potential in cancer treatment. This study offers novel insights how natural peptides can influence NK cell cytotoxicity. These pre-clinical findings hold significant promise for advancing current immunotherapeutic approaches.

Gene-guided identifications of a structure-chimeric cyclotide viphi I from Viola philippica: Potential functions against cadmium and nematodes.

The cyclic peptides, cyclotides, are identified mostly with 29-31-aa (amino acid residues) but rarely with ≥ 34-aa in plants. Viola philippica is a well-known medicinal plant but a rare metallophyte with cyclotides. A hypothesis was hence raised that the potential novel 34-aa cyclotide of Viola philippica would clearly broaden the structural and functional diversities of plant cyclotides. After homology-cloning the cyclotide precursor gene of VpCP5, a 34-aa cyclotide (viphi I) was identified to be larger than 22 other known cyclotides in V. philippica. It had a chimeric primary structure, due to its unusual loop structures (8 residues in loop 2 and 6 residues in loop 5) and aa composition (3 E and 5 R), by using phylogenetic analyses and an in-house cyclotide analysis tool, CyExcel_V1. A plasmid pCYC-viphi_I and a lab-used recombinant process were specially constructed for preparing viphi I. Typically, 0.12 or 0.25 mg ml-1 co-exposed viphi I could significantly remain cell activities with elevating Cd2+-exposed doses from 10-8 to 10-6 mol l-1 in MCF7 cells. In the model nematode Caenorhabditis elegans, IC50 values of viphi I to inhibit adult ratios and to induce death ratios, were 184.7 and 585.9 µg ml-1, respectively; the median lifespan of adult worms decreased from 14 to 2 d at viphi I doses ranging from 0.05 to 2 mg ml-1. Taken together, the newly identified viphi I exhibits functional potentials against cadmium and nematodes, providing new insights into structural and functional diversity of chimeric cyclotides in plants.

In Silico Analysis of Natural Plant-Derived Cyclotides with Antifungal Activity against Pathogenic Fungi.

BACKGROUND: Fungal infections in plants, animals, and humans are widespread across the world. Limited classes of antifungal drugs to treat fungal infections and loss of drug efficacy due to rapidly evolving fungal strains pose a challenge in the agriculture and health sectors. Hence, the search for a new class of antifungal agents is imperative. Cyclotides are cyclic plant peptides with multiple bioactivities, including antifungal activity. They have six conserved cysteine residues forming three disulfide linkages (CI-CIV, CII-CV, CIII-CVI) that establish a Cyclic Cystine Knot (CCK) structure, making them extremely resistant to chemical, enzymatic, and thermal attacks. AIM: This in silico analysis of natural, plant-derived cyclotides aimed to assess the parameters that can assist and hasten the process of selecting the cyclotides with potent antifungal activity and prioritize them for in vivo/ in vitro experiments. OBJECTIVE: The objective of this study was to conduct in silico studies to compare the physicochemical parameters, sequence diversity, surface structures, and membrane-cyclotide interactions of experimentally screened (from literature survey) potent (MIC ≤ 20 µM) and non-potent (MIC > 20 µM) cyclotides for antifungal activity. METHODOLOGY: Cyclotide sequences assessed for antifungal activity were retrieved from the database (Cybase). Various online and offline tools were used for sequence-based studies, such as physicochemical parameters, sequence diversity, and neighbor-joining trees. Structure-based studies involving surface structure analysis and membrane-cyclotide interaction were also carried out. All investigations were conducted in silico. RESULTS: Physicochemical parameter values, viz. isoelectric point, net charge, and the number of basic amino acids, were significantly higher in potent cyclotides compared to non-potent cyclotides. The surface structure of potent cyclotides showed a larger hydrophobic patch with a higher number of hydrophobic amino acids. Furthermore, the membrane-cyclotide interaction studies of potent cyclotides revealed lower transfer free energy (ΔG transfer) and higher penetration depth into fungal membranes, indicating higher binding stability and membrane-disruption ability. CONCLUSION: These in silico studies can be applied for rapidly identifying putatively potent antifungal cyclotides for in vivo and in vitro experiments, which will ultimately be relevant in the agriculture and pharmaceutical sectors.

Cyclotides: The next generation in biopesticide development for eco-friendly agriculture.

Chemical pesticides remain the predominant method for pest management in numerous countries. Given the current landscape of agriculture, the development of biopesticides has become increasingly crucial. The strategy empowers farmers to efficiently manage pests and diseases, while prioritizing minimal adverse effects on the environment and human health, hence fostering sustainable management. In recent years, there has been a growing interest and optimism surrounding the utilization of peptide biopesticides for crop protection. These sustainable and environmentally friendly substances have been recognized as viable alternatives to synthetic pesticides due to their outstanding environmental compatibility and efficacy. Numerous studies have been conducted to synthesize and identify peptides that exhibit activity against significant plant pathogens. One of the peptide classes is cyclotides, which are cyclic cysteine-rich peptides renowned for their wide range of sequences and functions. In this review, we conducted a comprehensive analysis of cyclotides, focusing on their structural attributes, developmental history, significant biological functions in crop protection, techniques for identification and investigation, and the application of biotechnology to enhance cyclotide synthesis. The objective is to emphasize the considerable potential of cyclotides as the next generation of plant protection agents on the global scale.

Effective Strategies in Designing Chitosan-hyaluronic Acid Nanocarriers: From Synthesis to Drug Delivery Towards Chemotherapy.

The biomedical field faces an ongoing challenge in developing more effective anti-cancer medication due to the significant burden that cancer poses on human health. Extensive research has been conducted on the utilization of natural polysaccharides in nanomedicine owing to their properties of biocompatibility, biodegradability, non-immunogenicity, and non-toxicity. These characteristics make them a potent drug delivery system for cancer therapy. The chitosan hyaluronic acid nanoparticle (CSHANp) system, consisting of chitosan and hyaluronic acid nanoparticles, has exhibited considerable potential as a nanocarrier for various cancer drugs, rendering it one of the most auspicious systems presently accessible. The CSHANps demonstrate remarkable drug loading capacity, precise control over drug release, and exceptional selectivity towards cancer cells. These properties enhance the therapeutic effectiveness against cancerous cells. This article aims to provide a comprehensive analysis of CSHANp, focusing on its characteristics, production techniques, applications, and future prospects.

Cyclotides derived from Viola dalatensis Gagnep: A novel approach for enrichment and evaluation of antimicrobial activity.

Cyclotides, plant-derived cysteine-rich peptides, exhibit a wide range of beneficial biological activities and possess exceptional structural stability. Cyclotides are commonly distributed throughout the Violaceae family. Viola dalatensis Gagnep, a Vietnamese species, has not been well studied, especially for cyclotides. This pioneering research explores cyclotides from V. dalatensis as antimicrobials. This study used a novel approach to enhance cyclotides after extraction. The approach combined 30% ammonium sulfate salt precipitation and RP-HPLC. A comprehensive analysis was performed to ascertain the overall protein content, flavonoids content, polyphenol content, and free radical scavenging capacity of compounds derived from V. dalatensis. Six known cyclotides were sequenced utilizing MS tandem. Semi-purified cyclotide mixtures (M1, M2, and M3) exhibited antibacterial efficacy against Bacillus subtilis (inhibitory diameters: 19.67-23.50 mm), Pseudomonas aeruginosa (22.17-23.50 mm), and Aspergillus flavus (14.67-21.33 mm). The enriched cyclotide precipitate from the stem extract demonstrated a minimum inhibitory concentration (MIC) of 0.08 mg/mL against P. aeruginosa, showcasing significant antibacterial effectiveness compared to the stem extract (MIC: 12.50 mg/mL). Considerable advancements have been achieved in the realm of cyclotides, specifically in their application as antimicrobial agents.

Molecular dynamics simulations support a preference of cyclotide kalata B1 for phosphatidylethanolamine phospholipids.

Kalata B1 (kB1), a naturally occurring cyclotide has been shown experimentally to bind lipid membranes that contain phosphatidylethanolamine (PE) phospholipids. Here, molecular dynamics simulations were used to explore its interaction with two phospholipids, palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), and a heterogeneous membrane comprising POPC/POPE (90:10), to understand the basis for the selectivity of kB1 towards PE phospholipids. The simulations showed that in the presence of only 10 % POPE lipid, kB1 forms a stable binding complex with membrane bilayers. An ionic interaction between the E7 carboxylate group of kB1 and the ammonium group of PE headgroups consistently initiates binding of kB1 to the membrane. Additionally, stable noncovalent interactions such as hydrogen bonding (E7, T8, V10, G11, T13 and N15), cation-π (W23), and CH-π (W23) interactions between specific residues of kB1 and the lipid membrane play an important role in stabilizing the binding. These findings are consistent with a structure-activity relationship study on kB1 where lysine mutagenesis on the bioactive and hydrophobic faces of the peptide abolished membrane-dependent bioactivities. In summary, our simulations suggest the importance of residue E7 (in the bioactive face) in enabling kB1 to recognize and bind selectively to PE-containing phospholipids bilayers through ionic and hydrogen bonding interactions, and of W23 (in the hydrophobic face) for the association and insertion of kB1 into the lipid bilayer through cation-π and CH-π interactions. Overall, this work enhances our understanding of the molecular basis of the membrane binding and bioactivity of this prototypic cyclotide.

Scanning mutagenesis identifies residues that improve the long-term stability and insecticidal activity of cyclotide kalata B1.

Cyclotides are plant-derived disulfide-rich cyclic peptides that have a natural function in plant defense and potential for use as agricultural pesticides. Because of their highly constrained topology, they are highly resistant to thermal, chemical, or enzymatic degradation. However, the stability of cyclotides at alkaline pH for incubation times of longer than a few days is poorly studied but important since these conditions could be encountered in the environment, during storage or field application as insecticides. In this study, kalata B1 (kB1), the prototypical cyclotide, was engineered to improve its long-term stability and retain its insecticidal activity via point mutations. We found that substituting either Asn29 or Gly1 to lysine or leucine increased the stability of kB1 by twofold when incubated in an alkaline buffer (pH = 9.0) for 7 days, while retaining its insecticidal activity. In addition, when Gly1 was replaced with lysine or leucine, the mutants could be cyclized using an asparaginyl endopeptidase, in vitro with a yield of ∼90% within 5 min. These results demonstrate the potential to manufacture kB1 mutants with increased stability and insecticidal activity recombinantly or in planta. Overall, the discovery of mutants of kB1 that have enhanced stability could be useful in leading to longer term activity in the field as bioinsecticides.

On chip microfluidic separation of cyclotides.

Cyclotides as a cyclic peptide produced by different groups of plants have been a very attractive field of research due to their exceptional properties in biological activities and drug design applications. The importance of cyclotides as new biological activities from nature caused to attract researchers to develop new separation systems. Recent growth and development on chip-based technology for separation and bioassay especially for anticancer having sparklingly advantages comparison with common traditional methods. In this study, the microfluidic separation of Vigno 1-5 cyclotides extracted from Viola ignobilis by using polar and nonpolar forces as a liquid-liquid interaction was investigated through modified microfluidic chips and then the results were compared with a traditional counterpart technique of high-performance liquid chromatography (HPLC). The traditional process of separating cyclotides from plants is a costly and time-consuming procedure. The scientific novelty of this study is to accelerate the separation of cyclotides using modified microfluidic chips with low cost and high efficiency. The results revealed that a novel and simple microfluidic chip concept is an effective approach for separating the Vigno groups in the violet extract. We believe that the concept could potentially be utilized for further drug development process especially for anticancer studies by coupling bioassay chips as online procedures via reducing in time and cost compared with traditional offline methods.

Lipidation of a bioactive cyclotide-based CXCR4 antagonist greatly improves its pharmacokinetic profile in vivo.

The CXCR4 chemokine is a key molecular regulator of many biological functions controlling leukocyte functions during inflammation and immunity, and during embryonic development. Overexpression of CXCR4 is also associated with many types of cancer where its activation promotes angiogenesis, tumor growth/survival, and metastasis. In addition, CXCR4 is involved in HIV replication, working as a co-receptor for viral entry, making CXCR4 a very attractive target for developing novel therapeutic agents. Here we report the pharmacokinetic profile in rats of a potent CXCR4 antagonist cyclotide, MCo-CVX-5c, previously developed in our group that displayed a remarkable in vivo resistance to biological degradation in serum. This bioactive cyclotide, however, was rapidly eliminated through renal clearance. Several lipidated versions of cyclotide MCo-CVX-5c showed a significant increase in the half-life when compared to the unlipidated form. The palmitoylated version of cyclotide MCo-CVX-5c displayed similar CXCR4 antagonistic activity as the unlipidated cyclotide, while the cyclotide modified with octadecanedioic (18-oxo-octadecanoic) acid exhibited a remarkable decrease in its ability to antagonize CXCR4. Similar results were also obtained when tested for its ability to inhibit growth in two cancer cell lines and HIV infection in cells. These results show that the half-life of cyclotides can be improved by lipidation although it can also affect their biological activity depending on the lipid employed.

Peptide modulators of cell migration: Overview, applications and future development.

Cell migration is a key physiological process in the development and homeostasis of multicellular organisms; errors in this complex system can trigger the development of cancer or inflammatory disorders. Therefore, modulating cell migration provides opportunities for drug discovery. Peptides are gaining importance on the global therapeutics market, given their unique properties compared with established small-molecule drugs or biologics. In this review, we identified over 470 peptides modulating cell migration and analyzed their characteristics. Over 95% of these peptides are in the discovery or preclinical stage, because the transition of peptide hits into drug leads often results in a bottleneck in the development process. We summarize chemical strategies in (pre-)clinical development to enhance drug-like properties of bioactive peptides.

Plant-based production of an orally active cyclotide for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.

Notes on the Treatment of Charged Particles for Studying Cyclotide/Membrane Interactions with Dissipative Particle Dynamics.

Different charge treatment approaches are examined for cyclotide-induced plasma membrane disruption by lipid extraction studied with dissipative particle dynamics. A pure Coulomb approach with truncated forces tuned to avoid individual strong ion pairing still reveals hidden statistical pairing effects that may lead to artificial membrane stabilization or distortion of cyclotide activity depending on the cyclotide's charge state. While qualitative behavior is not affected in an apparent manner, more sensitive quantitative evaluations can be systematically biased. The findings suggest a charge smearing of point charges by an adequate charge distribution. For large mesoscopic simulation boxes, approximations for the Ewald sum to account for mirror charges due to periodic boundary conditions are of negligible influence.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead, and an orally active analgesic is demonstrated.

Mutagenesis of bracelet cyclotide hyen D reveals functionally and structurally critical residues for membrane binding and cytotoxicity.

Cyclotides have a wide range of bioactivities relevant for agricultural and pharmaceutical applications. This large family of naturally occurring macrocyclic peptides is divided into three subfamilies, with the bracelet subfamily being the largest and comprising the most potent cyclotides reported to date. However, attempts to harness the natural bioactivities of bracelet cyclotides and engineer-optimized analogs have been hindered by a lack of understanding of the structural and functional role of their constituent residues, which has been challenging because bracelet cyclotides are difficult to produce synthetically. We recently established a facile strategy to make the I11L mutant of cyclotide hyen D that is as active as the parent peptide, enabling the subsequent production of a series of variants. In the current study, we report an alanine mutagenesis structure-activity study of [I11L] hyen D to probe the role of individual residues on peptide folding using analytical chromatography, on molecular function using surface plasmon resonance, and on therapeutic potential using cytotoxicity assays. We found that Glu-6 and Thr-15 are critical for maintaining the structure of bracelet cyclotides and that hydrophobic residues in loops 2 and 3 are essential for membrane binding and cytotoxic activity, findings that are distinct from the structural and functional characteristics determined for other cyclotide subfamilies. In conclusion, this is the first report of a mutagenesis scan conducted on a bracelet cyclotide, offering insights into their function and supporting future efforts to engineer bracelet cyclotides for biotechnological applications.

Scalable and Efficient In Planta Biosynthesis of Sunflower Trypsin Inhibitor-1 (SFTI) Peptide Therapeutics.

Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide which has been effectively employed as a scaffold for engineering a range of peptide therapeutic candidates. Typically, synthesis of SFTI-1-based therapeutics is performed via solid-phase peptide synthesis and native chemical ligation, with significant financial and environmental costs associated. In planta synthesis of SFTI-1 based therapeutics serves as a greener approach for environmentally sustainable production. Here, we detail the methods for the transient expression, production, and purification of SFTI-1-based therapeutic peptides in Nicotiana benthamiana using a scalable and high-throughput approach. We demonstrate that a prerequisite for this is the co-expression of specialized asparaginyl endopeptidases (AEPs) that perform the backbone cyclization of SFTI-1. In our founding study, we were able to achieve in planta yields of a plasmin inhibitor SFTI-1 peptide at yields of ~60 µg/g of dried plant material.

Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens.

According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to >8-fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.

Plant derived cyclic peptides.

Cyclic peptides are widespread throughout the plant kingdom, and display diverse sequences, structures and bioactivities. The potential applications attributed to these peptides and their unusual biosynthesis has captivated the attention of researchers for many years. Several gene sequences for plant cyclic peptides have been discovered over the last two decades but it is only recently that we are beginning to understand the intricacies associated with their biosynthesis. Recent studies have focussed on three main classes of plant derived cyclic peptides, namely orbitides, SFTI related peptides and cyclotides. In this mini-review, we discuss the expansion of the known sequence and structural diversity in these families, insights into the enzymes involved in the biosynthesis, the exciting applications which includes a cyclotide currently in clinical trials for the treatment of multiple sclerosis, and new production methods that are being developed to realise the potential of plant cyclic peptides as pharmaceutical or agricultural agents.

Linking molecular evolution to molecular grafting.

Molecular grafting is a strategy for the engineering of molecular scaffolds into new functional agents, such as next-generation therapeutics. Despite its wide use, studies so far have focused almost exclusively on demonstrating its utility rather than understanding the factors that lead to either poor or successful grafting outcomes. Here, we examine protein evolution and identify parallels between the natural process of protein functional diversification and the artificial process of molecular grafting. We discuss features of natural proteins that are correlated to innovability-the capacity to acquire new functions-and describe their implications to molecular grafting scaffolds. Disulfide-rich peptides are used as exemplars because they are particularly promising scaffolds onto which new functions can be grafted. This article provides a perspective on why some scaffolds are more suitable for grafting than others, identifying opportunities on how molecular grafting might be improved.

Topoly: Python package to analyze topology of polymers.

The increasing role of topology in (bio)physical properties of matter creates a need for an efficient method of detecting the topology of a (bio)polymer. However, the existing tools allow one to classify only the simplest knots and cannot be used in automated sample analysis. To answer this need, we created the Topoly Python package. This package enables the distinguishing of knots, slipknots, links and spatial graphs through the calculation of different topological polynomial invariants. It also enables one to create the minimal spanning surface on a given loop, e.g. to detect a lasso motif or to generate random closed polymers. It is capable of reading various file formats, including PDB. The extensive documentation along with test cases and the simplicity of the Python programming language make it a very simple to use yet powerful tool, suitable even for inexperienced users. Topoly can be obtained from https://topoly.cent.uw.edu.pl.