[Advances on cyclopeptides of plants].

Cyclopeptides isolated from a variety of plants are a class of cyclic nitrogen-containing compounds, and they are primarily formed by peptide bonds between amino acids, generally containing 2 to 37 L-configuration encoded or non-encoded amino acid residues. Cyclopeptides have significant values in scientific research as natural small-molecule metabolites produced by plants. The available studies have revealed that such natural products are ubiquitous in plants, which mainly include cyclic dipeptides, cyclic tetrapeptides, cyclic pentapeptides, cyclic hexapeptides, cyclic heptapeptides, cyclic octapeptides, cyclic nonapeptides, and cyclic decapeptides. Among them, cyclic dipeptides, cyclic hexapeptides, and cyclic octapeptides are the major active compounds. It has been reported that plant cyclopeptides have novel and unique chemical structures. They possess diverse pharmacological activities, such as antineoplastic, antimicrobial, antimalarial, anti-inflammatory, and antiviral activities. This paper summarizes the research achievements of plant cyclopeptides since 2006, aiming to provide theoretical reference for the research and application of plant cyclopeptides in medicine, health, and agriculture fields.

Regulation mechanism and bioactivity characteristic of surfactin homologues with C14 and C15 fatty acid chains.

BACKGROUND: Surfactin, a green lipopeptide bio-surfactant, exhibits excellent surface, hemolytic, antibacterial, and emulsifying activities. However, a lack of clear understanding of the synthesis regulation mechanism of surfactin homologue components has hindered the customized production of surfactin products with different biological activities. RESULTS: In this study, exogenous valine and 2-methylbutyric acid supplementation significantly facilitated the production of C14-C15 surfactin proportions (up to 75% or more), with a positive correlation between the homologue proportion and fortified concentration. Subsequently, the branched-chain amino acid degradation pathway and the glutamate synthesis pathway are identified as critical pathways in regulating C14-C15 surfactin synthesis by transcriptome analysis. Overexpression of genes bkdAB and glnA resulted in a 1.4-fold and 1.3-fold increase in C14 surfactin, respectively. Finally, the C14-rich surfactin was observed to significantly enhance emulsification activity, achieving an EI24 exceeding 60% against hexadecane, while simultaneously reducing hemolytic activity. Conversely, the C15-rich surfactin demonstrated an increase in both hemolytic and antibacterial activities. CONCLUSION: This study presents the first evidence of a potential connection between surfactin homologue synthesis and the conversion of glutamate and glutamine, providing a theoretical basis for targeting the synthesis regulation and structure-activity relationships of surfactin and other lipopeptide compounds.

Non-oxidized and oxidized flaxseed orbitides differently induce HepG2 cell apoptosis: involvement of cellular uptake and membrane death receptor DR4.

BACKGROUND: Flaxseed orbitides have health-promoting properties, particularly potent anti-cancer activity. However, flaxseed orbitides containing a methionine structure, such as [1-9-NαC]-linusorb B2 (CLB), are easily oxidized to sulfoxide ([1-9-NαC],[1-Rs,Ss-MetO]-linusorb-B2 (CLC)) and sulfone ([1-9-NαC], [1-MetO]-linusorb B2 (CLK)), with CLC having less anti-cancer ability than CLB. It is unclear why oxidized flaxseed orbitides are less effective against cancer than non-oxidized flaxseed orbitide. RESULTS: Non-oxidized ([1-9-NαC]-linusorb-B3 (CLA) and CLB) and oxidized (CLC and CLK) flaxseed orbitides were found to significantly upregulate the levels of pro-apoptotic proteins, including Bax/Bcl-2, CytoC, caspase-3, and caspase-8, in a dose-dependent manner, with non-oxidized flaxseed orbitides being more effective than oxidized flaxseed orbitides. Mechanically, the cellular absorption of non-oxidized flaxseed orbitides was higher than that of oxidized flaxseed orbitides. Moreover, the significant fluorescence quenching of DR4 protein by flaxseed orbitides (especially non-oxidized orbitides) indicated the formation of a DR4-orbitide complex. Molecular docking demonstrated that non-oxidized orbitides could easily dock into the active cavity of DR4 protein. Further blocking DR4 significantly reduced the ability of non-oxidized flaxseed orbitides to stimulate caspase-3 expression, whereas oxidized flaxseed orbitides retained this ability. CONCLUSION: Non-oxidized flaxseed orbitides are more effective against cancer than oxidized flaxseed orbitides due to higher cellular uptake and activation of the DR4-mediated death receptor signaling pathway. © 2024 Society of Chemical Industry.

Applications of biaryl cyclization in the synthesis of cyclic enkephalin analogs with a highly restricted flexibility.

A series of 10 cyclic, biaryl analogs of enkephalin, with Tyr or Phe residues at positions 1 and 4, were synthesized according to the Miyaura borylation and Suzuki coupling methodology. Biaryl bridges formed by side chains of the two aromatic amino acid residues are of the meta-meta, meta-para, para-meta, and para-para configuration. Conformational properties of the peptides were studied by CD and NMR. CD studies allowed only to compare conformations of individual peptides while NMR investigations followed by XPLOR calculations provided detailed information on their conformation. Reliability of the XPLOR calculations was confirmed by quantum chemical ones performed for one of the analogs. No intramolecular hydrogen bonds were found in all the peptides. They are folded and adopt the type IV ß-turn conformation. Due to a large steric strain, the aromatic carbon atoms forming the biaryl bond are distinctly pyramidalized. Seven of the peptides were tested in vitro for their affinity for the µ-opioid receptor.

Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Asparaginyl Endopeptidase-Mediated Peptide Cyclization for Phage Display.

We report here an enzymatic strategy for asparaginyl endopeptidase-mediated peptide cyclization. Incorporation of chloroacetyl groups into the recognition sequence of OaAEP1 enabled intramolecular cyclization with Cys residues. Combining this strategy and phage display, we identified nanomolar macrocyclic peptide ligands targeting TEAD4. One of the bicyclic peptides binds to TEAD4 with a KD value of 139 nM, 16 times lower than its linear analogue, demonstrating the utility of this platform in discovering high-affinity macrocyclic peptide ligands.

Membrane Permeability in a Large Macrocyclic Peptide Driven by a Saddle-Shaped Conformation.

The effort to modulate challenging protein targets has stimulated interest in ligands that are larger and more complex than typical small-molecule drugs. While combinatorial techniques such as mRNA display routinely produce high-affinity macrocyclic peptides against classically undruggable targets, poor membrane permeability has limited their use toward primarily extracellular targets. Understanding the passive membrane permeability of macrocyclic peptides would, in principle, improve our ability to design libraries whose leads can be more readily optimized against intracellular targets. Here, we investigate the permeabilities of over 200 macrocyclic 10-mers using the thioether cyclization motif commonly found in mRNA display macrocycle libraries. We identified the optimal lipophilicity range for achieving permeability in thioether-cyclized 10-mer cyclic peptide-peptoid hybrid scaffolds and showed that permeability could be maintained upon extensive permutation in the backbone. In one case, changing a single amino acid from d-Pro to d-NMe-Ala, representing the loss of a single methylene group in the side chain, resulted in a highly permeable scaffold in which the low-dielectric conformation shifted from the canonical cross-beta geometry of the parent compounds into a novel saddle-shaped fold in which all four backbone NH groups were sequestered from the solvent. This work provides an example by which pre-existing physicochemical knowledge of a scaffold can benefit the design of macrocyclic peptide mRNA display libraries, pointing toward an approach for biasing libraries toward permeability by design. Moreover, the compounds described herein are a further demonstration that geometrically diverse, highly permeable scaffolds exist well beyond conventional drug-like chemical space.

Alternative Linkage Chemistries in the Chemoenzymatic Synthesis of Microviridin-Based Cyclic Peptides.

Engineering biosynthetic pathways to ribosomally synthesized and post-translationally modified peptides (RiPPs) offers several advantages for both in vivo and in vitro applications. Here we probe the ability of peptide cyclases to generate trimacrocycle microviridin analogs with non-native cross-links. The results demonstrate that diverse chemistries are tolerated by macrocyclases in the ATP-grasp family and allow for the construction of unique cyclic peptide architectures that retain protease inhibition activity. In addition, cocomplex structures of analogs bound to a model protease were determined, illustrating how changes in functional groups maintain peptide conformation and target binding.

Modular design of cyclic peptide - polymer conjugate nanotubes for delivery and tunable release of anti-cancer drug compounds.

High aspect-ratio nanomaterials have recently emerged as promising drug delivery vehicles due to evidence of strong cellular association and prolonged in vivo circulation times. Cyclic peptide - polymer conjugate nanotubes are excellent candidates due to their elongated morphology, their supramolecular composition and high degree of pliability due to the versatility in manipulating amino acid sequence and polymer type. In this work, we explore the use of a nanotube structure on which a potent anti-cancer drug, camptothecin, is attached alongside hydrophilic or amphiphilic RAFT polymers, which shield the cargo. We show that subtle modifications to the cleavable linker type and polymer architecture have a dramatic influence over the rate of drug release in biological conditions. In vitro studies revealed that multiple cancer cell lines in 2D and 3D models responded effectively to the nanotube treatment, and analogous fluorescently labelled materials revealed key mechanistic information regarding the degree of cellular uptake and intracellular fate. Importantly, the ability to instruct specific drug release profiles indicates a potential for these nanomaterials as vectors which can provide sustained drug concentrations for a maximal therapeutic effect.

Design of cyclic peptides as novel inhibitors of ICOS/ICOSL interaction.

Compared to small molecules and antibodies, cyclic peptides exhibit unique biochemical and therapeutic attributes in the realm of pharmaceutical applications. The interaction between the inducible costimulator (ICOS) and its ligand (ICOSL) plays a key role in T-cell differentiation and activation. ICOS/ICOSL inhibition results in a reduction in the promotion of immunosuppressive regulatory T cells (Tregs) in both hematologic malignancies and solid tumors. Herein, we implement the computational cPEPmatch approach to design the first examples of cyclic peptides that inhibit ICOS/ICOSL interaction. The top cyclic peptide from our approach possessed an IC50 value of 1.87 ± 0.15 µM as an ICOS/ICOSL inhibitor and exhibited excellent in vitro pharmacokinetic properties as a drug candidate. Our work will lay the groundwork for future endeavors in cancer drug discovery, with the goal of developing cyclic peptides that target the ICOS/ICOSL interaction.

Antimicrobial and Anti-inflammatory Cyclic Tetrapeptides from the Co-cultures of Two Marine-Derived Fungi.

Violaceotides B-E (1-4), four new cyclic tetrapeptides, along with seven known compounds, were identified from the sponge-associated Aspergillus insulicola IMB18-072 co-cultivated with the marine-derived Alternaria angustiovoidea IMB20-805. Their structures were elucidated by extensive analysis of spectroscopic data, including HRESIMS, 1D and 2D NMR, and MS/MS data. The absolute configurations were determined by the advanced Marfey's method. Compounds 2, 3, and violaceotide A (5) displayed selective antimicrobial activities against the aquatic pathogenic bacteria Edwardsiella tarda and E. ictaluri. In addition, compounds 1-5 showed inhibitory activities against the LPS-induced expression of the inflammatory mediator IL-6 in RAW264.7 cells at a concentration of 10 µM.

Discovery and Characterization of a Bicyclic Peptide (Bicycle) Binder to Thymic Stromal Lymphopoietin.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein-protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

The Role of Attractive Non-Covalent Interactions in Peptide Macrocyclization.

The efficiency of macrocyclization reactions relies on the appropriate conformational preorganization of a linear precursor, ensuring that reactive ends are in spatial proximity prior to ring closure. Traditional peptide cyclization approaches that reduce the extent of terminal ion pairing often disfavor cyclization-conducive conformations and can lead to undesired cyclodimerization or oligomerization side reactions, particularly when they are performed without high dilution. To address this challenge, synthetic strategies that leverage attractive noncovalent interactions, such as zwitterionic attraction between chain termini during macrocyclization, offer a potential solution by reducing the entropic penalty associated with linear peptides adopting precyclization conformations. In this study, we investigate the role of (N-isocyanoimino)triphenylphosphorane (Pinc) in facilitating the cyclization of linear peptides into conformationally rigid macrocycles. The observed moderate diastereoselectivity is consistent with the preferential Si-facial addition of Pinc, where the isocyanide adds to the E-iminium ion on the same face as the l-proline amide group. The resulting peptide chain reveals that the activated phosphonium ylide of Pinc brings the reactive ends close together, promoting cyclization by enclosing the carboxylate within the interior of the pentapeptide and preventing the formation of byproducts. For shorter peptides with modified peptide backbones, the cyclization mechanism and outcome are redirected, as nucleophilic motifs such as thiazole and imidazole can covalently trap nitrilium intermediates. The isolation of the intermediate in the unproductive macrocyclization pathway, along with nuclear magnetic resonance and density functional theory studies, provides insights into heterocycle-dependent selectivity. The Pinc-driven macrocyclization process has generated diverse collections of cyclic molecules, and our models offer a comprehensive understanding of observed trends, facilitating the development of other heterocycle-forming macrocyclization reactions.

Cyclic Peptides for Drug Development.

Cyclic peptides are fascinating molecules abundantly found in nature and exploited as molecular format for drug development as well as other applications, ranging from research tools to food additives. Advances in peptide technologies made over many years through improved methods for synthesis and drug development have resulted in a steady stream of new drugs, with an average of around one cyclic peptide drug approved per year. Powerful technologies for screening random peptide libraries, and de novo generating ligands, have enabled the development of cyclic peptide drugs independent of naturally derived molecules and now offer virtually unlimited development opportunities. In this review, we feature therapeutically relevant cyclic peptides derived from nature and discuss the unique properties of cyclic peptides, the enormous technological advances in peptide ligand development in recent years, and current challenges and opportunities for developing cyclic peptides that address unmet medical needs.

Promiscuity of Omphalotin A Biosynthetic Enzymes Allows de novo Production of Non-Natural Multiply Backbone N-Methylated Peptide Macrocycles in Yeast.

Multiple backbone N-methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N-methylation and macrocyclization occur as part of both non-ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N-methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α-N-methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high-performance liquid chromatography, coupled with tandem mass spectrometry (HPLC-MS/MS). Our results demonstrate the successful production of fifteen non-natural omphalotin-derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non-natural multiply backbone N-methylated peptide macrocycles.

Molecular design and systematic optimization of a halogen-bonding system between the asthma interleukin-5 receptor and its cyclic peptide ligand.

Human interleukin-5 (IL-5) functions as an important pro-inflammatory factor by binding to its specific receptor, IL-5Rα, which has been implicated in the pathogenesis of asthma. Previously, a disulfide-bonded cyclic peptide AF17121 obtained from random library screening and sequence variation was found to competitively disrupt the cognate IL-5Rα/IL-5 interaction with moderate potency. In this study, the crystal complex of IL-5Rα with AF17121 was investigated at structural and energetic levels. It is revealed that the side-chain indole moiety of the AF17121 Trp5 residue is a potential site for a stem putative halogen bond (X-bond) with IL-5Rα, which is just located within the key 3 EXXR6 motif region recognized specifically by IL-5Rα. We systematically examined four halogen substitution types at five positions of the indole moiety; QM/MM calculations theoretically unraveled that only halogenations at 5 and 6 positions can form effective X-bonds with the side-chain hydroxyl oxygen of the IL-5Rα Thr21 residue and the backbone carbonyl oxygen of Ala66 residue, respectively. Binding assays observed that I-substitution at the 5 position and Br-substitution at the 6 position can result in two potent halogenated peptides, [5I]AF17121 and [6Br]AF17121, which are improved by 1.6-fold and 3.5-fold relative to the native AF17121, respectively. 5I/6Br-double substitution, resulting in [5I/6Br]AF17121, can further enhance the peptide affinity by 7.5-fold. Structural analysis revealed that the X-bond stemming from 6Br-substitution is also involved in an orthogonal interaction system with a H-bond; they share a common backbone carbonyl oxygen acceptor of IL-5Rα Ala66 residue and exhibit a significant synergistic effect between them.

P450-Modified Multicyclic Cyclophane-Containing Ribosomally Synthesized and Post-Translationally Modified Peptides.

Cyclic peptides with cyclophane linkers are an attractive compound type owing to the fine-tuned rigid three-dimensional structures and unusual biophysical features. Cytochrome P450 enzymes are capable of catalyzing not only the C-C and C-O oxidative coupling reactions found in vancomycin and other nonribosomal peptides (NRPs), but they also exhibit novel catalytic activities to generate cyclic ribosomally synthesized and post-translationally modified peptides (RiPPs) through cyclophane linkage. To discover more P450-modified multicyclic RiPPs, we set out to find cryptic and unknown P450-modified RiPP biosynthetic gene clusters (BGCs) through genome mining. Synergized bioinformatic analysis reveals that P450-modified RiPP BGCs are broadly distributed in bacteria and can be classified into 11 classes. Focusing on two classes of P450-modified RiPP BGCs where precursor peptides contain multiple conserved aromatic amino acid residues, we characterized 11 novel P450-modified multicyclic RiPPs with different cyclophane linkers through heterologous expression. Further mutation of the key ring-forming residues and combinatorial biosynthesis study revealed the order of bond formation and the specificity of P450s. This study reveals the functional diversity of P450 enzymes involved in the cyclophane-containing RiPPs and indicates that P450 enzymes are promising tools for rapidly obtaining structurally diverse cyclic peptide derivatives.

Rapid, traceless and facile peptide cyclization enabled by tetrazine-thiol exchange.

Cyclic peptides offer many advantages compared to their linear counterparts, including prolonged stability within the biological environment and enhanced binding affinity. Typically, peptides are cyclized by forming an amide bond, either on-resin or in solution, through extensive use of orthogonal protecting groups or chemoselective ligation strategies, respectively. Here, we show that the chemoselective tetrazine-thiol exchange is a powerful tool for rapid in situ cyclization of peptides without the need for additional activation reagents or extensive protecting group reshuffling. The reaction between N-terminal sulfide-bearing unsymmetric tetrazines and internal cysteines occurs spontaneously within a mildly acidic environment (pH 6.5) and is of traceless nature. The rapidly available unsymmetric sulfide tetrazine building blocks can be incorporated on resin using standard solid-phase peptide synthesis protocols and are orthogonal to trifluoroacetic acid cleavage conditions. The cyclized peptides display high stability, even when incubated with a large excess of free thiols. Due to its traceless and mild nature, we expect that the tetrazine-thiol exchange will be of high value for the in situ formation of cyclic peptide libraries, thus being applicable in drug discovery and development.

Molecular chameleons in drug discovery.

Molecular chameleons possess a flexibility that allows them to dynamically shield or expose polar functionalities in response to the properties of the environment. Although the concept of molecular chameleons was introduced already in 1970, interest in them has grown considerably since the 2010s, when drug discovery has focused to an increased extent on new chemical modalities. Such modalities include cyclic peptides, macrocycles and proteolysis-targeting chimeras, all of which reside in a chemical space far from that of traditional small-molecule drugs. Both cell permeability and aqueous solubility are required for the oral absorption of drugs. Engineering these properties, and potent target binding, into the larger new modalities is a more daunting task than for traditional small-molecule drugs. The ability of chameleons to adapt to different environments may be essential for success. In this Review, we provide both general and theoretical insights into the realm of molecular chameleons. We discuss why chameleons have come into fashion and provide a do-it-yourself toolbox for their design; we then provide a glimpse of how advanced in silico methods can support molecular chameleon design.

Exploring Cyclic Peptide Nanotube Stability Across Diverse Lipid Bilayers and Unveiling Water Transport Dynamics.

Cyclic Peptide Nanotubes (CPNTs) have emerged as compelling candidates for various applications, particularly as nanochannels within lipid bilayers. In this study, the stability of two CPNTs, namely 8 × [(Cys-Gly-Met-Gly)2] and 8 × [(Gly-Leu)4], are comprehensively investigated across different lipid bilayers, including 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), a mixed model membrane (POPE/POPG), and a realistic yeast model membrane. The results demonstrate that both CPNTs maintain their tubular structures in all lipid bilayers, with [(Cys-Gly-Met-Gly)2] showing increased stability over an extended period in these lipid membranes. The insertion of CPNTs shows negligible impact on lipid bilayer properties, including area per lipid, volume per lipid, and bilayer thickness. The study demonstrates that the CPNT preserves its two-line water movement pattern within all the lipid membranes, reaffirming their potential as water channels. The MSD curves further reveal that the dynamics of water molecules inside the nanotube are similar for all the bilayer systems with minor differences that arise due to different lipid environments.