Tricyclic Peptide Research Articles

Structural and biochemical studies of an iterative ribosomal peptide macrocyclase.

Microviridins, tricyclic peptide natural products originally isolated from cyanobacteria, function as inhibitors of diverse serine-type proteases. Here we report the structure and biochemical characterization of AMdnB, a unique iterative macrocyclase involved in a microviridin biosynthetic pathway from Anabaena sp. PCC 7120. The ATP-dependent cyclase, along with the homologous AMdnC, introduce up to nine macrocyclizations on three distinct core regions of a precursor peptide, AMdnA. The results presented here provide structural and mechanistic insight into the iterative chemistry of AMdnB. In vitro AMdnB-catalyzed cyclization reactions demonstrate the synthesis of the two predicted tricyclic products from a multi-core precursor peptide substrate, consistent with a distributive mode of catalysis. The X-ray structure of AMdnB shows a structural motif common to ATP-grasp cyclases involved in RiPPs biosynthesis. Additionally, comparison with the noniterative MdnB allows insight into the structural basis for the iterative chemistry. Overall, the presented results provide insight into the general mechanism of iterative enzymes in ribosomally synthesized and post-translationally modified peptide biosynthetic pathways.

A Series of Novel, Highly Potent, and Orally Bioavailable Next-Generation Tricyclic Peptide PCSK9 Inhibitors.

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) is a key regulator of plasma LDL-cholesterol (LDL-C) and a clinically validated target for the treatment of hypercholesterolemia and coronary artery disease. Starting from second-generation lead structures such as 2, we were able to refine these structures to obtain extremely potent bi- and tricyclic PCSK9 inhibitor peptides. Optimized molecules such as 44 demonstrated sufficient oral bioavailability to maintain therapeutic levels in rats and cynomolgus monkeys after dosing with an enabled formulation. We demonstrated target engagement and LDL lowering in cynomolgus monkeys essentially identical to those observed with the clinically approved, parenterally dosed antibodies. These molecules represent the first report of highly potent and orally bioavailable macrocyclic peptide PCSK9 inhibitors with overall profiles favorable for potential development as once-daily oral lipid-lowering agents. In this manuscript, we detail the design criteria and multiparameter optimization of this novel series of PCSK9 inhibitors.

Heterologous expression of a cryptic gene cluster from Marinomonas fungiae affords a novel tricyclic peptide marinomonasin

The ω-ester-containing peptides (OEPs) are a group of ribosomally synthesized and post-translationally modified peptides (RiPPs). The biosynthetic gene clusters of ω-ester-containing peptides commonly include ATP-grasp ligase coding genes and are distributed over the genomes of a wide variety of bacteria. A new biosynthetic gene cluster of ω-ester-containing peptides was found in the genome sequence of the marine proteobacterium Marinomonas fungiae. Heterologous production of a new tricyclic peptide named marinomonasin was accomplished using the biosynthetic gene cluster in Escherichia coli expression host strain BL21(DE3). By ESI-MS and NMR experiments, the structure of marinomonasin was determined to be a tricyclic peptide 18 amino acids in length with one ester and two isopeptide bonds in the molecule. The bridging patterns of the three intramolecular bonds were determined by the interpretation of HMBC and NOESY data. The bridging pattern of marinomonasin was unprecedented in the ω-ester-containing peptide group. The results indicated that the ATP-grasp ligase for the production of marinomonasin was a novel enzyme possessing bifunctional activity to form one ester and two isopeptide bonds. KEY POINTS: • New tricyclic peptide marinomonasin was heterologously produced in Escherichia coli. • Marinomonasin contained one ester and two isopeptide bonds in the molecule. • The bridging pattern of intramolecular bonds was novel.

Click-Free Synthesis of a Multivalent Tricyclic Peptide as a Molecular Transporter.

The cellular delivery of cell-impermeable and water-insoluble molecules remains an ongoing challenge to overcome. Previously, we reported amphipathic cyclic peptides c[WR]4 and c[WR]5 consisting of alternate arginine and tryptophan residues as nuclear-targeting molecular transporters. These peptides contain an optimal balance of positive charge and hydrophobicity, which is required for interactions with the phospholipid bilayer to facilitate their application as a drug delivery system. To further optimize them, we synthesized and evaluated a multivalent tricyclic peptide as an efficient molecular transporter. The monomeric cyclic peptide building blocks were synthesized using Fmoc/tBu solid-phase chemistry and cyclization in the solution and conjugated with each other through an amide bond to afford the tricyclic peptide, which demonstrated modest antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli (E. coli) with a minimum inhibitory concentration (MIC) of 64–128 µg/mL. The tricyclic peptide was found to be nontoxic up to 30 µM in the breast cancer cell lines (MDA-MB-231). The presence of tricyclic peptide enhanced cellular uptakes of fluorescently-labeled phosphopeptide (F’-GpYEEI, 18-fold), anti-HIV drugs (lamivudine (F’-3TC), emtricitabine (F’-FTC), and stavudine (F’-d4T), 1.7–12-fold), and siRNA (3.3-fold) in the MDA-MB-231 cell lines.

Design and Synthesis of Disulfide-Rich Peptides with Orthogonal Disulfide Pairing Motifs.

Disulfide-rich peptides (DRPs) are a class of peptides that are constrained through two or more disulfide bonds. Though natural DRPs have been extensively exploited for developing protein binders or potential therapeutics, their synthesis and re-engineering to bind new targets are not straightforward due to difficulties in handling the disulfide pairing problem. Rationally designed DRPs with an intrinsically orthogonal disulfide pairing propensity provide an alternative to the natural scaffolds for developing functional DRPs. Herein we report the use of tandem CXPen/PenXC motifs ((C) cysteine; (Pen) penicillamine; (X) any residue) for directing the oxidative folding of peptides. Diverse tricyclic peptides were designed and synthesized by varying the pattern of C/Pen residues and incorporating a tandem CXPen/PenXC motif into peptides. The folding of these peptides was determined primarily by C/Pen patterns and tolerated to sequence manipulations. The applicability of the designed C/Pen-DRPs was demonstrated by designing protein binders using an epitope grafting strategy. This study thus demonstrates the potential of using orthogonal disulfide pairing to design DRP scaffolds with new structures and functions, which would greatly benefit the development of multicyclic peptide ligands and therapeutics.

Heterologous expression of a cryptic gene cluster from Grimontia marina affords a novel tricyclic peptide grimoviridin.

Microviridins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that have been isolated from a wide variety of cyanobacterial strains. There are similar gene clusters of RiPPs distributed in the genomes of bacteria belonging to the phyla Proteobacteria and Bacteroidetes. A cryptic gene cluster for the production of microviridin-type peptide was found in the genome of the marine γ-Proteobacterium Grimontia marina. Heterologous production of new microviridin-type peptide named grimoviridin was accomplished in Escherichia coli using the biosynthetic gene cluster of G. marina. The structure of grimoviridin was determined by analysis of MS and NMR data. Grimoviridin contained one isopeptide and two ester bonds, which had exactly the same bridging pattern as other microviridin-type peptides. The absolute stereochemistries of constituent amino acids were determined to be all L-forms by modified Marfey's method. Grimoviridin showed potent inhibitory activity against trypsin with an IC50 value of 238nM. This is the first report of heterologous production of microviridin-type peptide using a biosynthetic gene cluster from a Proteobacterium. Key points • Heterologous production afforded new microviridin-type peptide named grimoviridin. • This is the first report of microviridin-type peptide from proteobacterial origin. • Grimoviridin showed potent inhibitory activity against trypsin.

Enzymatic Cascade To Evaluate the Tricyclization of Glycopeptide Antibiotic Precursor Peptides as a Prequel to Biosynthetic Redesign.

Natural products are the greatest source of antimicrobial agents, although their structural complexity often renders synthetic production and diversification of key classes impractical. One pertinent example is the glycopeptide antibiotics (GPAs), which are highly challenging to synthesize due to their heavily cross-linked structures. Here, we report an optimized method that generates >75% tricyclic peptides from synthetic precursors in order to explore the acceptance of novel GPA precursor peptides by these key existent biosynthetic enzymes.

Unlocking the Spatial Control of Secondary Metabolism Uncovers Hidden Natural Product Diversity in Nostoc punctiforme.

Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.

Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation

AbstractBicyclic and tricyclic peptides have emerged as promising candidates for the development of protein binders and new therapeutics. However, convenient and efficient strategies that can generate topologically controlled bicyclic and tricyclic peptide scaffolds from fully‐unprotected peptides are still much in demand, particularly for those amenable to the design of biosynthetic libraries. In this work, we report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and tricyclic peptides. This work thus lays the foundation for developing peptide libraries with controlled topology of multicyclic scaffolds for in vitro display techniques.

Chemical and Ribosomal Synthesis of Topologically Controlled Bicyclic and Tricyclic Peptide Scaffolds Primed by Selenoether Formation.

Bicyclic and tricyclic peptides have emerged as promising candidates for the development of protein binders and new therapeutics. However, convenient and efficient strategies that can generate topologically controlled bicyclic and tricyclic peptide scaffolds from fully-unprotected peptides are still much in demand, particularly for those amenable to the design of biosynthetic libraries. In this work, we report a reliable chemical and ribosomal synthesis of topologically controlled bicyclic and tricyclic peptide scaffolds. Our strategy involves the combination of selenoether cyclization followed by disulfide or thioether cyclization, yielding desirable bicyclic and tricyclic peptides. This work thus lays the foundation for developing peptide libraries with controlled topology of multicyclic scaffolds for in vitro display techniques.

Inside Cover: General and Facile Route to Isomerically Pure Tricyclic Peptides Based on Templated Tandem CLIPS/CuAAC Cyclizations (Angew. Chem. Int. Ed. 2/2018)

Inside Cover: General and Facile Route to Isomerically Pure Tricyclic Peptides Based on Templated Tandem CLIPS/CuAAC Cyclizations (Angew. Chem. Int. Ed. 2/2018)

General and Facile Route to Isomerically Pure Tricyclic Peptides Based on Templated Tandem CLIPS/CuAAC Cyclizations

AbstractWe report a one‐pot ligation/cyclization technology for the rapid and clean conversion of linear peptides into tricyclic peptides that is based on using tetravalent scaffolds containing two benzyl bromide and two alkyne moieties. These react via CLIPS/CuAAC reactions with cysteines and azides in the peptide. Flexibility in the scaffolds is key to the formation of isomerically pure products as the flexible scaffolds T41 and T42 mostly promote the formation of single isomeric tricycles while the rigid scaffolds T43 and T44 do not yield clean products. There seems to be no limitation to the number and types of amino acids present as 18 canonical amino acids were successfully implemented. We also observed that azides at the peptide termini and cysteine residues in the center gave better results than compounds with the functional groups placed the other way round.

General and Facile Route to Isomerically Pure Tricyclic Peptides Based on Templated Tandem CLIPS/CuAAC Cyclizations.

We report a one-pot ligation/cyclization technology for the rapid and clean conversion of linear peptides into tricyclic peptides that is based on using tetravalent scaffolds containing two benzyl bromide and two alkyne moieties. These react via CLIPS/CuAAC reactions with cysteines and azides in the peptide. Flexibility in the scaffolds is key to the formation of isomerically pure products as the flexible scaffolds T41 and T42 mostly promote the formation of single isomeric tricycles while the rigid scaffolds T43 and T44 do not yield clean products. There seems to be no limitation to the number and types of amino acids present as 18 canonical amino acids were successfully implemented. We also observed that azides at the peptide termini and cysteine residues in the center gave better results than compounds with the functional groups placed the other way round.

Omniligase‐1: A Powerful Tool for Peptide Head‐to‐Tail Cyclization

AbstractStrategies for the efficient synthesis of peptide macrocycles have been a long‐standing goal. In this paper, we demonstrate the use of the peptide ligase termed omniligase‐1 as a versatile and broadly applicable enzymatic tool for peptide cyclization. Several head‐to‐tail (multi)cyclic peptides have been synthesized, including the cyclotide MCoTI‐II. Cyclization and oxidative folding of the cyclotide MCoTI‐II were efficiently performed in a one‐pot reaction on a 1‐gram scale. The native cyclotide was isolated and the correct disulfide bonding pattern was confirmed by NMR structure determination. Furthermore, compatibility of chemo‐enzymatic peptide synthesis (CEPS) using omniligase‐1 with methods such as chemical ligation of peptides onto scaffolds (CLIPS) was successfully demonstrated by synthesizing a kinase‐inhibitor derived tricyclic peptide. Our studies indicate that the minimal ring size for omniligase‐1 mediated cyclization is 11 amino acids, whereas the cyclization of peptides longer than 12 amino acids proceeds with remarkable efficiency. In addition, several macrocycles containing non‐peptidic backbones (e.g., polyethylene glycol), isopeptide bonds (amino acid side‐chain attachment) as well as d‐amino acids could be efficiently cyclized.magnified image

Precisely Regulated and Efficient Locking of Linear Peptides into Stable Multicyclic Topologies through a One‐Pot Reaction

AbstractWe report the discovery of a small phenyl molecule with four isosteric thiolate‐reactive groups of sequentially varied reactivity. This molecule was exploited in combination with cysteine/penicillamine thiolates of different nucleophilic reactivity for precisely regulated and efficient locking (PROP‐locking) of linear peptides into multicyclic topologies through a one‐pot reaction. The PROP‐locking relies on multistep and sequential thiolate/fluorine nucleophilic substitutions, which is not only rapid but highly specific, thus enabling rapid locking of peptides with high amino acid diversities without protecting groups. Several tricyclic peptide templates and bioactive peptides were designed and synthesized using the PROP‐locking strategy. We believe that tricyclic peptides precisely locked through stable thioether bonds should be promising structurally constrained scaffolds for developing potential therapeutics and target ligands.

Precisely Regulated and Efficient Locking of Linear Peptides into Stable Multicyclic Topologies through a One-Pot Reaction.

We report the discovery of a small phenyl molecule with four isosteric thiolate-reactive groups of sequentially varied reactivity. This molecule was exploited in combination with cysteine/penicillamine thiolates of different nucleophilic reactivity for precisely regulated and efficient locking (PROP-locking) of linear peptides into multicyclic topologies through a one-pot reaction. The PROP-locking relies on multistep and sequential thiolate/fluorine nucleophilic substitutions, which is not only rapid but highly specific, thus enabling rapid locking of peptides with high amino acid diversities without protecting groups. Several tricyclic peptide templates and bioactive peptides were designed and synthesized using the PROP-locking strategy. We believe that tricyclic peptides precisely locked through stable thioether bonds should be promising structurally constrained scaffolds for developing potential therapeutics and target ligands.

Oligopeptides in plant medicines cited in Chinese Pharmacopoeia

In total, 23 plant plant medicined containing oligopeptides were cited in Chinese Pharmacopoeia (1 part) of 2015 version including Rubia cordifolia, Linum usitatissimum, Aster tataricus, Psammosilene tunicoides, Pseudostellaria heterophylla, Stellaria dichotoma, Vaccaria segetalis, Dianthus superbus, Celosia argentea, Lycii Cortex, Citrus medica, C. aurantium, Panax ginseng, Parmx notoginseng, Schisandra chinensis, Sparganium stoloniferum, Euryale ferox, Ophiopogon japonicas, Pinellia ternate, Achyranthes bidentata, Physalis alkekengi, Polygonatum odoratum, and Leonuri Fructus. There were 187 oligopeptides in plant medicines above as reported. Oligopeptides consisted mainly of linear peptides and cyclic peptides. The linear peptides included dipeptides, tripeptides and pentapeptides, and cyclic peptides included cyclic, bicyclic and tricyclic peptides. The number of residues of single cyclic peptides ranged from two to twelve. Bicyclic peptides were isolated mainly from R. cordifolia and C. argentea. Modern pharmacological study showed that oligopeptides had many pharmacological effects, including antitumor, anticoagulant, antibacterial, immune suppression and so on.

Synthesis of fused tricyclic peptides using a reprogrammed translation system and chemical modification

Here we report a unique method of ribosomally synthesizing fused tricyclic peptides. Flexizyme-assisted in vitro translation of a linear peptide with the N-terminal chloroacetyl group and four downstream cysteines followed by the addition of 1,3,5-tris(bromomethyl)benzene results in selective production of the fused tricyclic peptide. This technology can be used for the ribosomal synthesis of fused tricyclic peptide libraries for the in vitro selection of bioactive peptides with tricyclic topology.

Synthesis of Tricyclic Isoindole Cyclic Peptide Based on Photoinduced Single Electron Trasfer Reaction

Exploratory photochemical studies on phthalimido-polypeptides 1a~1h in which phthalimides worked as light absorbance acceptors and polypeptide served as electron donor centers have been conducted, leading to the development of synthetic methodology to construct tricyclic isoindole cyclopeptide mimetics. It was found that irradiation on 1a, 1b, 1d~1h in methanol led to efficient generation of tricyclic isoindole cyclic peptides 2a, 2b, 2d~2h, which may be through sequential single electron transfer (SET) pathway. The structures of new compounds were determined by NMR and HRMS spectroscopy.

Functional Analysis of Environmental DNA-Derived Microviridins Provides New Insights into the Diversity of the Tricyclic Peptide Family

Microviridins represent a unique family of ribosomally synthesized cage-like depsipeptides from cyanobacteria with potent protease-inhibitory activities. The natural diversity of these peptides is largely unexplored. Here, we describe two methodologies that were developed to functionally characterize cryptic microviridin gene clusters from metagenomic DNA. Environmental samples were collected and enriched from cyanobacterial freshwater blooms of different geographical origins containing predominantly Microcystis sp. Microviridins were produced either directly from fosmid clones or after insertion of environmental DNA-derived gene cassettes into a minimal expression platform in Escherichia coli. Three novel microviridin variants were isolated and tested against different serine-type proteases. The comparison of the bioactivity profiles of the new congeners allows deduction of further structure-function relationships for microviridins. Moreover, this study provides new insights into microviridin processing and gene cluster organization.