Cyclic Tetrapeptide Research Articles

Peptides inhibiting the assembly of monomeric human l-lactate dehydrogenase into catalytically active homotetramer decrease the synthesis of lactate in cultured cells.

The energetic metabolism of cancer cells relies on a substantial commitment of pyruvate to the catalytic action of lactate-generating dehydrogenases. This coupling mainly depends on lactate dehydrogenase A (LDH-A), which is overexpressed in different types of cancers, and therefore represents an appealing therapeutic target. Taking into account that the activity of LDHs is exclusively exerted by their tetrameric forms, it was recently shown that peptides perturbing the monomers-to-tetramer assembly inhibit human LDH-A (hLDH-A). However, to identify these peptides, tetrameric hLDH-A was transiently exposed to strongly acidic conditions inducing its dissociation into monomers, which were tested as a target for peptides at low pH. Nevertheless, the availability of native monomeric hLDH-A would allow performing similar screenings under physiological conditions. Here we report on the unprecedented isolation of recombinant monomeric hLDH-A at neutral pH, and on its use to identify peptides inhibiting the assembly of the tetrameric enzyme. Remarkably, the GQNGISDL octapeptide, mimicking the 296-303 portion of hLDH-A C-terminal region, was observed to effectively inhibit the target enzyme. Moreover, by dissecting the action of this octapeptide, the cGQND cyclic tetrapeptide was found to act as the parental compound. Furthermore, we performed assays using MCF7 and BxPC3 cultured cells, exclusively expressing hLDH-A and hLDH-B, respectively. By means of these assays we detected a selective action of linear and cyclic GQND tetrapeptides, inhibiting lactate secretion in MCF7 cells only. Overall, our observations suggest that peptides mimicking the C-terminal region of hLDH-A effectively interfere with protein-protein interactions responsible for the assembly of the tetrameric enzyme.

Micro-scale screening of genetically modified Fusarium fujikuroi strain extends the apicidin family

Apicidins are a class of naturally occurring cyclic tetrapeptides produced by few strains within the Fusarium genus. These secondary metabolites have gained significant attention due to their antiprotozoal activity through HDAC inhibition, thereby highlighting their potential for the treatment of malaria. Predominantly, apicidins have been isolated from Fusarium semitectum, offering a deep insight into the biosynthetic pathway responsible for their formation. A similar biosynthetic gene cluster has also been identified in the rice pathogenic fungus F. fujikuroi, leading the discovery of three additional apicidins through genetic manipulation. Routine mass spectrometric screening of these compound-producing strains revealed another metabolite structurally related to previously studied apicidins. By optimizing culture conditions and developing an effective isolation method, we obtained a highly pure substance, whose chemical structure was fully elucidated using NMR and HRMS fragmentation. Further studies were conducted to determine cytotoxicity, antimalarial activity, and HDAC inhibitory activity of this new secondary metabolite alongside the previously known apicidins. This work not only expands the apicidin class with a new member but also provides extensive insights and comparative analysis of apicidin-like substances produced by F. fujikuroi.Graphical

Influence of heterochirality on the structure, dynamics, biological properties of cyclic(PFPF) tetrapeptides obtained by solvent-free ball mill mechanosynthesis

Cyclic tetrapeptides c(Pro-Phe-Pro-Phe) obtained by the mechanosynthetic method using a ball mill were isolated in a pure stereochemical form as a homochiral system (all L-amino acids, sample A) and as a heterochiral system with D configuration at one of the stereogenic centers of Phe (sample B). The structure and stereochemistry of both samples were determined by X-ray diffraction studies of single crystals. In DMSO and acetonitrile, sample A exists as an equimolar mixture of two conformers, while only one is monitored for sample B. The conformational space and energetic preferences for possible conformers were calculated using DFT methods. The distinctly different conformational flexibility of the two samples was experimentally proven by Variable Temperature (VT) and 2D EXSY NMR measurements. Both samples were docked to histone deacetylase HDAC8. Cytotoxic studies proved that none of the tested cyclic peptide is toxic.

Total Synthesis of the Naturally Occurring Cyclic Tetrapeptide JM‐47and Analogues through Late‐Stage Functionalization of a Common Scaffold

AbstractA unified approach to access the naturally occurring cyclic tetrapeptide JM‐47 and some of its structural variants from a common macrocyclic scaffold has been developed. The key reactions involved a difficult macrocyclization to access the scaffold, and its late‐stage diversification by cross‐metathesis to access the natural product as well as analogues. The synthesis proceeded in good overall yield and high stereochemical integrity. It also confirms the structure and configuration assigned to JM‐47.

Synthesis and Functionalization of Azetidine-Containing Small Macrocyclic Peptides.

Cyclic peptides are increasingly important structures in drugs but their development can be impeded by difficulties associated with their synthesis. Here, we introduce the 3-aminoazetidine (3-AAz) subunit as a new turn-inducing element for the efficient synthesis of small head-to-tail cyclic peptides. Greatly improved cyclizations of tetra-, penta- and hexapeptides (28 examples) under standard reaction conditions are achieved by introduction of this element within the linear peptide precursor. Post-cyclization deprotection of the amino acid side chains with strong acid is realized without degradation of the strained four-membered azetidine. A special feature of this chemistry is that further late-stage modification of the resultant macrocyclic peptides can be achieved via the 3-AAz unit. This is done by: (i) chemoselective deprotection and substitution at the azetidine nitrogen, or by (ii) a click-based approach employing a 2-propynyl carbamate on the azetidine nitrogen. In this way, a range of dye and biotin tagged macrocycles are readily produced. Structural insights gained by XRD analysis of a cyclic tetrapeptide indicate that the azetidine ring encourages access to the less stable, all-trans conformation. Moreover, introduction of a 3-AAz into a representative cyclohexapeptide improves stability towards proteases compared to the homodetic macrocycle.

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.

A MassQL-Integrated Molecular Networking Approach for the Discovery and Substructure Annotation of Bioactive Cyclic Peptides.

The marine sponge-derived fungus Stachylidium bicolor 293 K04 is a prolific producer of specialized metabolites, including certain cyclic tetrapeptides called endolides, which are characterized by the presence of the unusual amino acid N-methyl-3-(3-furyl)-alanine. This rare feature can be used as bait to detect new endolide-like analogs through customized fragment pattern searches of tandem mass spectrometry data using the Mass Spec Query Language (MassQL). Here, we integrate endolide-specific MassQL queries with molecular networking to obtain substructural information guiding the targeted isolation and structure elucidation of the new proline-containing endolides E (1) and F (2). We showed that endolide F (but not E) is a moderate antagonist of the arginine vasopressin V1A receptor, a member of the G protein-coupled receptor superfamily.

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.

Synthesis and biological evaluation of nectriatide derivatives, potentiators of amphotericin B activity.

Nectriatide 1a, a naturally occurring cyclic tetrapeptide, has been reported to a potentiator of amphotericin B (AmB) activity. In order to elucidate its structure-activity relationships, we synthesized nectriatide derivatives with different amino acids in solution-phase synthesis and evaluated AmB-potentiating activity against Candida albicans. Among them, C-and N-terminal protected linear peptides were found to show the most potent AmB-potentiating activity.

Direct synthesis of β2,3‐amino acid from β‐hydroxycarbamides: Implication in the synthesis of azumamide analogs and conformational analysis

AbstractThe designed β2,3 unnatural amino acids have been synthesized from α‐methyl‐β‐hydroxycarbamides via direct azidation without obtaining eliminated products. Furthermore, these β2,3 amino acids were utilized to synthesize cyclic tetrapeptides (CTPs) as azumamide analogs. 2D‐NMR analysis revealed the compact secondary structure as a single conformer. Presence of intramolecular H‐bonding in CTPs was observed in this α3β2,3 architecture and displayed a seven‐membered γ‐turn structure.

Characterization of Arthropeptide B, an Antifungal Cyclic Tetrapeptide from Arthrobacter humicola.

Disease suppressive composts are known, yet little information on the potential role of specific microbial antagonist within are available. Arthrobacter humicola isolate M9-1A has been obtained from a compost prepared from marine residues and peat moss. The bacterium is a non-filamentous actinomycete with antagonistic activity against plant pathogenic fungi and oomycetes sharing its ecological niche in agri-food microecosystems. Our objective was to identify and characterize compounds with antifungal activity produced by A. humicola M9-1A. Arthrobacter humicola culture filtrates were tested for antifungal activity in vitro and in vivo and a bioassay-guided approach was used to identify potential chemical determinants of its observed activity against molds. The filtrates reduced the development of lesions of Alternaria rot on tomatoes and the ethyl acetate extract inhibited growth of Alternaria alternata. A compound, arthropeptide B [cyclo-(L-Leu, L-Phe, L-Ala, L-Tyr)], was purified from the ethyl acetate extract of the bacterium. Arthropeptide B is a new chemical structure reported for the first time and has shown antifungal activity against A. alternata spore germination and mycelial growth.

Cyclic Tetrapeptide Natural Product Asperterrestide A: Total Synthesis and Structural Revision

We have studied for the total synthesis of asperterrestide A, which is a cyclic tetrapeptide natural product reported to be composed of D-alanine, anthranilic acid, (2R,3S)-3-hydroxy-N-methylphenylalanine, and D-isoleucine or D-allo-isoleucine. We initially performed the total synthesis of the reported structure of asperterrestide A via solid-phase peptide synthesis and macrocyclization in solution. However, the spectral data of the synthetic (9R,25R)-derivative did not match those of the natural product. Then, we considered the true structure should be the (9S,25S)-stereoisomer because all NOE correlations predicted from its three-dimensional structures obtained by theoretical calculations were consistent with those observed in the natural product. Thus, the (9S,25S)-stereoisomer was synthesized through solution-phase synthesis because N-acylation of a polymer-supported (2S,3S)-3-hydroxy-N-methylphenylalanine derivative resulted in low yield. The (9S,25S)-stereoisomer was successfully synthesized and was found to be identical to the natural product. Therefore, the stereochemical configuration of asperterrestide A was corrected from (9R,25R) to (9S,25S). We also tested whether the DFT calculation of carbon chemical shifts can discriminate stereoisomers of cyclic tetrapeptides. After conformational analysis of four possible stereoisomers of asperterrestide A, Boltzmann-weighted carbon chemical shifts based on DFT calculations using Spartan’20 revealed a DP4 accuracy of 96%, which is sufficient to determine the stereochemical configuration among the stereoisomers. In addition, three 9-deoxy derivatives were synthesized and the cell growth inhibitory activity of the five synthetic compounds against human cancer cells (U937, MOLT-4, and A549) was evaluated. The results showed that the (9S,25S)-stereoisomer is as potent as asperterrestide A, whereas the (9R,25R)-derivative is not. The (9S,25S)-3-deoxy analogs were found to be equipotent to the natural product although the activity of the (9S,25R)-deoxy analog was lost.

Macrocyclic Octapeptide Binding and Inferences on Protein Substrate Binding to Histone Deacetylase 6

Histone deacetylases (HDACs) are essential for the regulation of myriad biological processes, and their aberrant function is implicated in cancer, neurodegeneration, and other diseases. The cytosolic isozyme HDAC6 is unique among the greater family of deacetylases in that it contains two catalytic domains, CD1 and CD2. HDAC6 CD2 is responsible for tubulin deacetylase and tau deacetylase activities, inhibition of which is a key goal as new therapeutic approaches are explored. Of particular interest as HDAC inhibitors are naturally occurring cyclic tetrapeptides such as Trapoxin A or HC Toxin, or the cyclic depsipeptides Largazole and Romidepsin. Even more intriguing are larger, computationally designed macrocyclic peptide inhibitors. Here, we report the 2.0 Å resolution crystal structure of HDAC6 CD2 complexed with macrocyclic octapeptide 1. Comparison with the previously reported structure of the complex with macrocyclic octapeptide 2 reveals that a potent thiolate-zinc interaction made by the unnatural amino acid (S)-2-amino-7-sulfanylheptanoic acid contributes to nanomolar inhibitory potency for each inhibitor. Apart from this zinc-binding residue, octapeptides adopt strikingly different overall conformations and make few direct hydrogen bonds with the protein. Intermolecular interactions are dominated by water-mediated hydrogen bonds; in essence, water molecules appear to cushion the enzyme-octapeptide interface. In view of the broad specificity observed for protein substrates of HDAC6 CD2, we suggest that the binding of macrocyclic octapeptides may mimic certain features of the binding of macromolecular protein substrates.

Conformation Based in silico Studies of Cyclic Tetrapeptides with βγ Fused Turns as Thrombin Inhibitors

AbstractThe enormous social and economic consequences of thrombotic diseases, as well as the significant dangers associated with present medications, need the development of more effective and safer anticoagulants. In silico structure‐based design was used to find new peptide‐based thrombin inhibitors and cyclic peptide‐based molecules are attracting wide attention in that regard. Here we have shown the synthesis, conformational and in silico analysis of tetrahydrofuran amino acid (TAA) based 15‐membered cyclic tetrapeptides (CTPs) containing guanidine side chain as thrombin inhibitors. Solution NMR conformational analysis suggests that the βγ fused structures of CTPs are retained in the core and there is no effect of guanidine functionality on the scaffold. Molecular docking results showed that these CTPs can inhibit thrombin protein by interacting with Asp189 residue and the core structure helps to retain the guanidine group inside the active site of protein without any alteration in the backbone H‐bonding. Overall, we have presented here the synthesis, conformational analysis of TAA containing cyclic tetrapeptides along with their docking study against thrombin protein. Further these results will help to design peptide based novel thrombin inhibitors.

Bulbiferamide, an Antitrypanosomal Hexapeptide Cyclized via an N-Acylindole Linkage from a Marine Obligate Microbulbifer.

UV absorption spectroscopy-guided fractionation of the culture extract of a marine obligate bacterium of the genus Microbulbifer yielded a novel cyclic hexapeptide, bulbiferamide (1). NMR spectroscopic and mass spectrometric analyses revealed the structure of 1 to be a cyclic tetrapeptide appending a ureido-bridged two amino acid unit. Notably, Trp is a junction residue, forming on one hand a very rare N-aminoacylated indole linkage for cyclization and on the other hand connecting the ureido-containing tail structure, which is an unprecedented way of configuring peptides. The component amino acids were determined to be l by the advanced Marfey's method. Compound 1 displayed growth inhibitory activity against Trypanosoma cruzi epimastigotes with an IC50 value of 4.1 μM, comparable to the currently approved drug benznidazole, while it was not cytotoxic to P388 murine leukemia cells at 100 μM.

Diastereoselective synthesis of cyclic tetrapeptide pseudoxylallemycin A illuminates the impact of base during macrolactamization.

Therapeutic agents with unique molecular structures and new mechanisms of action are needed to confront the phenomenon of multidrug resistance among bacteria. Pseudoxylallemycins, cyclic tetrapeptide (CTP) natural products, have exhibited modest antibiotic activity, but their synthesis has proven challenging. Inherent ring strain in CTPs decreases the rate of cyclization in lieu of polymerization and racemization pathways, which has resulted in previous syntheses describing mixtures of diastereomers containing predominantly an undesired epimer. We have optimized the cyclization step of pseudoxylallemycin A to favor production of the natural diastereomer; notably, variation of the base, temperature, and solvent with peptide coupling reagent propylphosphonic anhydride (T3P) afforded exquisite selectivity for the natural product in as high as 97 : 3 DR, and our conditions can provide the natural product in up to 32% overall yield through 8 steps. Employing weaker bases than those typically used in peptide coupling reactions led to the greatest improvement in diastereoselectivity, and these studies demonstrated that the identity of the amine base has enormous impact on the rate of C-terminal epimerization when T3P is used, a variable usually considered of lesser consequence when combined with typical amide coupling reagents. Toward fully characterizing pseudoxylallemycin stereoisomers, variable temperature NMR was described as a tool to more clearly analyze CTPs that exhibit multiple conformational states. These synthetic and spectroscopic insights were applied toward synthesizing several natural product analogues, and their antibacterial activity was examined using microdilution assays.

Quantitative characterization of filamentous fungal promoters on a single-cell resolution to discover cryptic natural products.

Characterization of filamentous fungal regulatory elements remains challenging because of time-consuming transformation technologies and limited quantitative methods. Here we established a method for quantitative assessment of filamentous fungal promoters based on flow cytometry detection of the superfolder green fluorescent protein at single-cell resolution. Using this quantitative method, we acquired a library of 93 native promoter elements from Aspergillus nidulans in a high-throughput format. The strengths of identified promoters covered a 37-fold range by flow cytometry. PzipA and PsltA were identified as the strongest promoters, which were 2.9- and 1.5-fold higher than that of the commonly used constitutive promoter PgpdA. Thus, we applied PzipA and PsltA to activate the silent nonribosomal peptide synthetase gene Afpes1 from Aspergillus fumigatus in its native host and the heterologous host A. nidulans. The metabolic products of Afpes1 were identified as new cyclic tetrapeptide derivatives, namely, fumiganins A and B. Our method provides an innovative strategy for natural product discovery in fungi.

Synthesis of L-cyclic tetrapeptides by backbone amide activation CyClick strategy

Cyclic tetrapeptides exhibit high cellular permeability and a wide range of biological properties and thus have gained great interest in the field of medicinal chemistry. We synthesized highly strained 12-membered head to tail cyclic peptides with varying reactive amino acids, without oligomerization using the exclusively intramolecular CyClick chemistry. This occurs by a two-step process involving the low-energy formation of a 15 atom-containing cyclic imine, followed by a chemoselective ring contraction of the peptide backbone generating a highly strained 12 atom-containing cyclic tetrapeptide. This reaction exhibited high substrate scope and generated head to tail cyclic tetrapeptides with varying amino acids at the N-terminus, showing chemoselectivity without the need for side group protection.

Structural and Biofunctional Insights into the Cyclo(Pro-Pro-Phe-Phe-) Scaffold from Experimental and In Silico Studies: Melanoma and Beyond.

Short peptides have great potential as safe and effective anticancer drug leads. Herein, the influence of short cyclic peptides containing the Pro-Pro-Phe-Phe sequence on patient-derived melanoma cells was investigated. Cyclic peptides such as cyclo(Leu-Ile-Ile-Leu-Val-Pro-Pro-Phe-Phe-), called CLA, and cyclo(Pro-homoPro-β3homoPhe-Phe-), called P11, exert the cytotoxic and the cytostatic effects in melanoma cells, respectively. CLA was the most active peptide as it reduced the viability of melanoma cells to 50% of control at about 10 µM, whereas P11 at about 40 µM after 48 h incubation. Interestingly, a linear derivative of P11 did not induce any effect in melanoma cells confirming previous studies showing that cyclic peptides exert better biological activity compared to their linear counterparts. According to in silico predictions, cyclic tetrapeptides show a better pharmacokinetic and toxic profile to humans than CLA. Notably, the spatial structure of those peptides containing synthetic amino acids has not been explored yet. In the Cambridge Structural Database, there is only one such cyclic tetrapeptide, cyclo((R)-β2homoPhe-D-Pro-Lys-Phe-), while in the Protein Data Bank—none. Therefore, we report the first crystal structure of cyclo(Pro-Pro-β3homoPhe-Phe-), denoted as 4B8M, a close analog of P11, which is crucial for drug discovery. Comparative molecular and supramolecular analysis of both structures was performed. The DFT findings revealed that 4B8M is well interpreted in the water solution. The results of complex Hirshfeld surface investigations on the cooperativity of interatomic contacts in terms of electrostatic and energetic features are provided. In short, the enrichment ratio revealed O…H/H…O and C…H/H…C as privileged intercontacts in the crystals in relation to basic and large supramolecular H-bonding synthon patterns. Furthermore, the ability of self-assemble 4B8M leading to a nanotubular structure is also discussed.

Antiviral Activity of a Cyclic Pro-Pro-β3-HoPhe-Phe Tetrapeptide against HSV-1 and HAdV-5.

The core of Cyclolinopeptide A (CLA, cyclo(LIILVPPFF)), responsible for its high immunosuppressive activity, contains a Pro-Pro-Phe-Phe sequence. A newly synthesized cyclic tetrapeptide, cyclo(Pro-Pro-β3-HoPhe-Phe) (denoted as 4B8M) bearing the active sequence of CLA, was recently shown to exhibit a wide array of anti-inflammatory properties in mouse models. In this investigation, we demonstrate that the peptide significantly inhibits the replication of human adenovirus C serotype 5 (HAdV-5) and Herpes simplex virus type-1 (HSV-1) in epithelial lung cell line A-549, applying Cidofovir and Acyclovir as reference drugs. Based on a previously established mechanism of its action, we propose that the peptide may inhibit virus replication by the induction of PGE2 acting via EP2/EP4 receptors in epithelial cells. In summary, we reveal a new, antiviral property of this anti-inflammatory peptide.