Cyclic Peptide Natural Products Research Articles

Synthesis and biological evaluation of echinomycin analogues as potential colon cancer agent

Colorectal cancer is the third most commonly diagnosed cancer and the second leading cause of cancer-related death, thus a novel chemotherapeutic agent for colon cancer therapy is needed. In this study, analogues of echinomycin, a cyclic peptide natural product with potent toxicity to several human cancer cell lines, were synthesized, and their biological activities against human colon cancer cells were investigated. Analogue 3 as well as 1 inhibit HIF-1α-mediated transcription. Notably, transcriptome analysis indicated that the cell cycle and its regulation were involved in the effects on cells treated with 3. Analogue 3 exhibited superior in vivo efficacy to echinomycin without significant toxicity in mouse xenograft model. The low dose of 3 needed to be efficacious in vivo is also noteworthy and our data suggest that 3 is an attractive and potentially novel agent for the treatment of colon cancer.

Use of a head-to-tail peptide cyclase to prepare hybrid RiPPs.

Cyclotides and lanthipeptides are cyclic peptide natural products with promising bioengineering potential. No peptides have been isolated that contain both structural motifs defining these two families, an N-to-C cyclised backbone and lanthionine linkages. We combined their biosynthetic machineries to produce hybrid structures that possess improved activity or stability, demonstrate how the AEP-1 plant cyclase can be utilised to complete the maturation of the sactipeptide subtilosin A, and present head-to-tail cyclisation of the glycocin sublancin. These studies show the plasticity of AEP-1 and its utilisation alongside other post-translational modifications.

Discovery and biosynthesis of tricyclic copper-binding ribosomal peptides containing histidine-to-butyrine crosslinks

Cyclic peptide natural products represent an important class of bioactive compounds and clinical drugs. Enzymatic side-chain macrocyclization of ribosomal peptides is a major strategy developed by nature to generate these chemotypes, as exemplified by the superfamily of ribosomally synthesized and post-translational modified peptides. Despite the diverse types of side-chain crosslinks in this superfamily, the participation of histidine residues is rare. Herein, we report the discovery and biosynthesis of bacteria-derived tricyclic lanthipeptide noursin, which is constrained by a tri amino acid labionin crosslink and an unprecedented histidine-to-butyrine crosslink, named histidinobutyrine. Noursin displays copper-binding ability that requires the histidinobutyrine crosslink and represents the first copper-binding lanthipeptide. A subgroup of lanthipeptide synthetases, named LanKCHbt, were identified to catalyze the formation of both the labionin and the histidinobutyrine crosslinks in precursor peptides and produce noursin-like compounds. The discovery of the histidinobutyrine-containing lanthipeptides expands the scope of post-translational modifications, structural diversity and bioactivity of ribosomally synthesized and post-translational modified peptides.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus.

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with backbone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from "drained" strains.

Momomycin, an Antiproliferative Cryptic Metabolite from the Oxytetracycline Producer Streptomyces rimosus

Natural products provide an important source of pharmaceuticals and chemical tools. Traditionally, assessment of unexplored microbial phyla has led to new natural products. However, with every new microbe, the number of orphan biosynthetic gene clusters (BGC) grows. As such, the more difficult proposition is finding new molecules from well-studied strains. Herein, we targeted Streptomyces rimosus, the widely-used oxytetracycline producer, for the discovery of new natural products. Using MALDI-MS-guided high-throughput elicitor screening (HiTES), we mapped the global secondary metabolome of S. rimosus and structurally characterized products of three cryptic BGCs, including momomycin, an unusual cyclic peptide natural product with back-bone modifications and several non-canonical amino acids. We elucidated important aspects of its biosynthesis and evaluated its bioactivity. Our studies showcase HiTES as an effective approach for unearthing new chemical matter from “drained” strains

A New Amino Acid for Improving Permeability and Solubility in Macrocyclic Peptides through Side Chain-to-Backbone Hydrogen Bonding.

Despite the notoriously poor membrane permeability of peptides, many cyclic peptide natural products show high passive membrane permeability and potently inhibit a variety of "undruggable" intracellular targets. A major impediment to the design of cyclic peptides with good permeability is the high desolvation energy associated with the peptide backbone amide NH groups. While several strategies have been proposed to mitigate this deleterious effect, only few studies have used polar side chains to sequester backbone NH groups. We investigated the ability of N,N-pyrrolidinylglutamine (Pye), whose side chain contains a powerful hydrogen-bond-accepting C═O amide group but no hydrogen-bond donors, to sequester exposed backbone NH groups in a series of cyclic hexapeptide diastereomers. Analyses revealed that specific Leu-to-Pye substitutions conferred dramatic improvements in aqueous solubility and permeability in a scaffold- and position-dependent manner. Therefore, this approach offers a complementary tool for improving membrane permeability and solubility in cyclic peptides.

The tryptophan connection: cyclic peptide natural products linked via the tryptophan side chain.

Covering: from 1938 up to March 2021The electron-rich indole side chain of tryptophan is a versatile substrate for peptide modification. Upon the action of various cyclases, the tryptophan side chain may be linked to a nearby amino acid residue, opening the door to a diverse range of cyclic peptide natural products. These compounds exhibit a wide array of biological activity and possess fascinating molecular architectures, which have made them popular targets for total synthesis studies. This review examines the isolation and bioactivity of tryptophan-linked cyclic peptide natural products, along with a discussion of their first total synthesis, and biosynthesis where this has been studied.

Serine/Threonine Ligation and Cysteine/Penicillamine Ligation.

Serine/threonine ligation (STL) and cysteine/penicillamine ligation (CPL) are highly chemo- and regioselective reactions between unprotected peptides with C-terminus salicylaldehyde esters and unprotected peptides with N-terminus serine/threonine or cysteine/penicillamine, which serve as powerful tools for cyclic peptide natural product and chemical protein synthesis. Herein, we introduce the preparation of C-terminal peptide salicylaldehyde esters, serine/threonine ligation, cysteine/penicillamine ligation, and subsequent acidolysis.

Identifying the Cellular Target of Cordyheptapeptide A and Synthetic Derivatives.

Cordyheptapeptide A is a lipophilic cyclic peptide from the prized Cordyceps fungal genus that shows potent cytotoxicity in multiple cancer cell lines. To better understand the bioactivity and physicochemical properties of cordyheptapeptide A with the ultimate goal of identifying its cellular target, we developed a solid-phase synthesis of this multiply N-methylated cyclic heptapeptide which enabled rapid access to both side chain- and backbone-modified derivatives. Removal of one of the backbone amide N-methyl (N-Me) groups maintained bioactivity, while membrane permeability was also preserved due to the formation of a new intramolecular hydrogen bond in a low dielectric solvent. Based on its cytotoxicity profile in the NCI-60 cell line panel, as well as its phenotype in a microscopy-based cytological assay, we hypothesized that cordyheptapeptide was acting on cells as a protein synthesis inhibitor. Further studies revealed the molecular target of cordyheptapeptide A to be the eukaryotic translation elongation factor 1A (eEF1A), a target shared by other lipophilic cyclic peptide natural products. This work offers a strategy to study and improve cyclic peptide natural products while highlighting the ability of these lipophilic compounds to effectively inhibit intracellular disease targets.

Post-translational formation of strained cyclophanes in bacteria.

Cyclic peptide natural products have served as important drug molecules, with several examples used clinically. Enzymatic or chemical macrocyclization is the key transformation for constructing these chemotypes. Methods to generate new and diverse cyclic peptide scaffolds enabling the modular and predictable synthesis of peptide libraries are desirable in drug discovery platforms. Here we identify a suite of post-translational modifying enzymes from bacteria that install single or multiple strained cyclophane macrocycles. The crosslinking occurs on three-residue motifs that include tryptophan or phenylalanine to form indole- or phenyl-bridged cyclophanes. The macrocycles display restricted rotation of the aromatic ring and induce planar chirality in the asymmetric indole bridge. The biosynthetic gene clusters originate from a broad range of bacteria derived from marine, terrestrial and human microbiomes. Three-residue cyclophane-forming enzymes define a new and significant natural product family and occupy a distinct region in sequence-function space.

Direct Peptide Cyclization and One-Pot Modification Using the MeDbz Linker.

The one-pot synthesis and modification of cyclic peptides through a self-cleaving on-resin protocol is described. We apply Dawson's MeDbz linker to achieve direct intramolecular peptide cyclization by thioesterification followed by S → N acyl shift. This native chemical ligation approach requires no activating additive and allows direct modification of the crude cyclic peptides in one-pot. The strategy was applied to synthesize 5 cyclic peptide natural products of varying ring size. Finally, one-pot modifications include desulfurization, fluorophore conjugation, and intramolecular disulfide formation.

Serine/Threonine Ligation: Origin, Mechanistic Aspects, and Applications.

Synthetic proteins are expected to go beyond the boundary of recombinant DNA expression systems by being flexibly installed with site-specific natural or unnatural modification structures during synthesis. To enable protein chemical synthesis, peptide ligations provide effective strategies to assemble short peptide fragments obtained from solid-phase peptide synthesis (SPPS) into long peptides and proteins. In this regard, chemoselective peptide ligation represents a simple but powerful transformation realizing selective amide formation between the C-terminus and N-terminus of two side-chain-unprotected peptide fragments. These reactions are highly chemo- and regioselective to tolerate the side-chain functionalities present on the unprotected peptides, highly reactive to work with millmolar or submillimolar concentrations of the substrates, and operationally simple with mild conditions and accessible building blocks. This Account focuses on our work in the development of serine/threonine ligation (STL), which originates from a chemoselective reaction between an unprotected peptide with a C-terminal salicylaldehyde (SAL) ester and another unprotected peptide with an N-terminal serine or threonine residue. Mechanistically, STL involves imine capture, 5- endo-trig ring-chain tautomerization, O-to- N [1,5] acyl transfer to afford the N, O-benzylidene acetal-linked peptide, and acidolysis to regenerate the Xaa-Ser/Thr linkage (where Xaa is the amino acid) at the ligation site. The high abundance of serine and threonine residues (12.7%) in naturally occurring proteins and the good compatibility of STL with various C-terminal residues provide multiple choices for ligation sites. The requisite peptide C-terminal SAL esters can be prepared from the peptide fragments obtained from both Fmoc-SPPS and Boc-SPPS through four available methods (a safety-catch strategy based on phenolysis, direct coupling, ozonolysis, and the n + 1 strategy). In the synthesis of proteins (e.g., ACYP enzyme, MUC1 glycopeptide 40-mer to 80-mer, interleukin 25, and HMGA1a with variable post-translational modification patterns), both C-to- N and N-to- C sequential STL strategies have been developed through selection of temporal N-terminal protecting groups and proper design of the switch-on/off C-terminal SAL ester surrogate, respectively. In the synthesis of cyclic peptide natural products (e.g., daptomycin, teixobactin, cyclomontanin B, yunnanin C) and their analogues, intramolecular head-to-tail STL has been implemented on linear peptide SAL ester precursors containing four to 10 amino acid residues with good efficiency and minimized oligomerization. As a thiol-independent chemoselective ligation complementary to native chemical ligation, STL provides an alternative tool for the chemical synthesis of homogeneous proteins with site-specific and structure-defined modifications and cyclic peptide natural products, which lays foundation for chemical biology and medicinal studies of those molecules with biological importance and therapeutic potential.

Stereochemistry Balances Cell Permeability and Solubility in the Naturally Derived Phepropeptin Cyclic Peptides.

Cyclic peptide (CP) natural products provide useful model systems for mapping "beyond-Rule-of-5" (bRo5) space. We identified the phepropeptins as natural product CPs with potential cell permeability. Synthesis of the phepropeptins and epimeric analogues revealed much more rapid cellular permeability for the natural stereochemical pattern. Despite being more cell permeable, the natural compounds exhibited similar aqueous solubility as the corresponding epimers, a phenomenon explained by solvent-dependent conformational flexibility among the natural compounds. When analyzing the polarity of the solution structures we found that neither the number of hydrogen bonds nor the total polar surface area accurately represents the solvation energies of the high and low dielectric conformations. This work adds to a growing number of natural CPs whose solvent-dependent conformational behavior allows for a balance between aqueous solubility and cell permeability, highlighting structural flexibility as an important consideration in the design of molecules in bRo5 chemical space.

多成分反応を用いた天然物合成と構造活性相関研究

Multicomponent reaction represented by Mannich reaction, Strecker reaction and Biginelli reaction has been applied to several natural product synthesis because a complexed framework of the natural product can be constructed by assembling more than three simple components at one step. It is also attractive from a medicinal chemical point of view because it enables us to supply a range of analogues to investigate a structure-activity relationship simply by changing components in the reaction. Here, we described our recent studies utilizing a multicomponent reaction in natural product-based medicinal chemistry. First topic is about total synthesis of syringolin A class of proteasome inhibitor and its structure-activity relationship study by intramolecular Ugi three-component reaction. We also synthesized cyclic peptide natural products (sandramycin, quinaldopeptin) and analogues and revealed their biological properties. Finally, design and synthesis of simplified caprazamycin analogues using aza-Prins-Ritter reaction was described.

Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex.

Cyclic peptide natural products have evolved to exploit diverse protein targets, many of which control essential cellular processes. Inspired by a series of cyclic peptides with partially elucidated structures, we designed synthetic variants of ternatin, a cytotoxic and anti-adipogenic natural product whose molecular mode of action was unknown. The new ternatin variants are cytotoxic toward cancer cells, with up to 500-fold greater potency than ternatin itself. Using a ternatin photo-affinity probe, we identify the translation elongation factor-1A ternary complex (eEF1A·GTP·aminoacyl-tRNA) as a specific target and demonstrate competitive binding by the unrelated natural products, didemnin and cytotrienin. Mutations in domain III of eEF1A prevent ternatin binding and confer resistance to its cytotoxic effects, implicating the adjacent hydrophobic surface as a functional hot spot for eEF1A modulation. We conclude that the eukaryotic elongation factor-1A and its ternary complex with GTP and aminoacyl-tRNA are common targets for the evolution of cytotoxic natural products.

Total Synthesis of Ustiloxin D Utilizing an Ammonia–Ugi Reaction

Total synthesis of the highly functionalized cyclic peptide natural product, ustiloxin D, has been achieved in a convergent manner. Our strategy incorporates an asymmetric allylic alkylation to construct the tert-alkyl aryl ether linkage between the dopa and isoleucine residues. The elaborated β-hydroxydopa derivative is rapidly converted to a linear tripeptide through an ammonia-Ugi reaction. Subsequent cyclization and global deprotection affords ustiloxin D in six steps from a known β-hydroxydopa derivative.

Interrogating cyclic peptide natural products from an ADME perspective: Beyond cyclosporine A.

Interrogating cyclic peptide natural products from an ADME perspective: Beyond cyclosporine A.

Beyond cyclosporine A: conformation-dependent passive membrane permeabilities of cyclic peptide natural products.

Many cyclic peptide natural products are larger and structurally more complex than conventional small molecule drugs. Although some molecules in this class are known to possess favorable pharmacokinetic properties, there have been few reports on the membrane permeabilities of cyclic peptide natural products. Here, we present the passive membrane permeabilities of 39 cyclic peptide natural products, and interpret the results using a computational permeability prediction algorithm based on their known or calculated 3D conformations. We found that the permeabilities of these compounds, measured in a parallel artificial membrane permeability assay, spanned a wide range and demonstrated the important influence of conformation on membrane permeability. These results will aid in the development of these compounds as a viable drug paradigm.

Probing the Physicochemical Boundaries of Cell Permeability and Oral Bioavailability in Lipophilic Macrocycles Inspired by Natural Products.

Cyclic peptide natural products contain a variety of conserved, nonproteinogenic structural elements such as d-amino acids and amide N-methylation. In addition, many cyclic peptides incorporate γ-amino acids and other elements derived from polyketide synthases. We hypothesized that the position and orientation of these extended backbone elements impact the ADME properties of these hybrid molecules, especially their ability to cross cell membranes and avoid metabolic degradation. Here we report the synthesis of cyclic hexapeptide diastereomers containing γ-amino acids (e.g., statines) and systematically investigate their structure-permeability relationships. These compounds were much more water-soluble and, in many cases, were both more membrane permeable and more stable to liver microsomes than a similar non-statine-containing derivative. Permeability correlated well with the extent of intramolecular hydrogen bonding observed in the solution structures determined in the low-dielectric solvent CDCl3, and one compound showed an oral bioavailability of 21% in rat. Thus, the incorporation of γ-amino acids offers a route to increase backbone diversity and improve ADME properties in cyclic peptide scaffolds.

Serine/threonine ligation for natural cyclic peptide syntheses.

Covering: 2000 to 2014Cyclic peptides are a class of abundant natural products, often exhibiting attractive biological activities. The challenges in the total synthesis of cyclic peptides lie in the preparation of unnatural amino acids if present and the peptide cyclization. Cyclization is an entropy-disfavoured process, with competition between intermolecular and intramolecular reactions. Biological methods can utilize the pre-organized conformation of the side chain unprotected peptide, which brings the reacting termini into proximity, while chemical synthesis requires protecting groups, often large-size and hydrophobic in nature. In this regard, performing peptide cyclization can be an arbitrary and trial-and-error practice. In this highlight, we discuss the application of chemoselective ligation-mediated peptide cyclization in the total synthesis of natural cyclic peptides.