RESUMEN
In this study, microcin J25, a potent antimicrobial lasso peptide that acts on Gram-negative bacteria, was subjected to a harsh treatment with a base in order to interrogate its stability and mechanism of action and explore its structure-activity relationship. Despite the high stability reported for this lasso peptide, the chemical treatment led to the detection of a new product. Structural studies revealed that this product retained the lasso topology, but showed no antimicrobial activity due to the epimerization of a key residue for the activity. Further microbiological assays also demonstrated that it showed a high synergistic effect with colistin.
Asunto(s)
Antiinfecciosos/química , Bacteriocinas/química , Antiinfecciosos/farmacología , Bacteriocinas/farmacología , Colistina/química , Colistina/farmacología , Sinergismo Farmacológico , Pruebas de Sensibilidad Microbiana , Estabilidad ProteicaRESUMEN
Lasso peptides are natural products belonging to the family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) and are defined by their unique topology. Even though lasso peptide biosynthetic gene clusters are found in many different kinds of bacteria, most of the hitherto studied lasso peptides were of proteobacterial or actinobacterial origin. Despite this, no E. coli-based production system has been reported for actinobacterial lasso peptides, while there are numerous examples of this for proteobacterial lasso peptides. Here, a heterologous production system of the lasso peptide chaxapeptin was established in E. coli. Chaxapeptin, originally isolated from Streptomyces leeuwenhoekii strain C58, is closely related to the lasso peptide sungsanpin (produced by a marine Streptomyces sp.) and shares its inhibitory activity against cell invasion by the human lung cancer cell line A549. Our production system not only allowed isolation of the mature lasso peptide outside of the native producer with a yield of 0.1 mg/L (compared to 0.7 mg/L from S. leeuwenhoekii) but also was used for a mutational study to identify residues in the precursor peptide that are important for biosynthesis. In addition to these experiments, the stability of chaxapeptin against thermal denaturation and proteases was assessed.
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Escherichia coli/genética , Péptidos Cíclicos/biosíntesis , Estabilidad de Medicamentos , Escherichia coli/metabolismo , Familia de Multigenes , Mutación , Péptidos Cíclicos/químicaRESUMEN
The chemical synthesis of a bicycle inspired by the natural lasso peptide sungsanpin using a combination of solid-phase and in-solution chemistries is described. The bicyclic-derived topoisomer was designed by introducing a covalent linkage between the ring and the loop, which allowed the tying of these two parts of the peptide, rendering the bicyclic structure. Several structural techniques, such as MS fragmentation, ion-mobility and NMR spectroscopic analysis were used to characterize the bicycle. Ion-mobility spectroscopy studies revealed that it showed lasso-like behavior. Its 3D structure was predicted on the basis of the NMR restraints. In addition, the high proteolytic and thermal stability of the bicycle potentially make it a suitable scaffold for epitope grafting.
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Compuestos Bicíclicos con Puentes/química , Péptidos/química , Compuestos Bicíclicos con Puentes/síntesis química , Modelos Moleculares , Péptidos/síntesis química , Conformación Proteica , Estabilidad ProteicaRESUMEN
The discovery and development of novel constrained peptides that combine the advantages of therapeutic proteins with those of small molecules has partially prompted the re-emergence of peptides as therapeutics. In this regard, lasso peptides are characterized by both the selectivity and potency of larger protein biologics but with no or low immunogenicity, and the stability and bioavailability of small molecules. Moreover, the diverse functionality of lasso peptides and their extraordinary stability against chemical, thermal and proteolytic degradation make them attractive candidates for drug discovery. However, the chemical synthesis of lasso peptides remains a challenge due to the difficulty in building and maintaining their threaded structure. From a therapeutic point of view, these small and constrained structures would provide a new paradigm in drug discovery.
RESUMEN
Cyclodepsipeptides are cyclic peptides in which at least one amide link on the backbone is replaced with an ester link. These natural products present a high structural diversity that corresponds to a broad range of biological activities. Therefore, they are very promising pharmaceutical candidates. Most of the cyclodepsipeptides have been isolated from marine organisms, but they can also originate from terrestrial sources. Within the family of cyclodepsipeptides, 'head-to-side-chain' cyclodepsipeptides have, in addition to the macrocyclic core closed by the ester bond, an arm terminated with a polyketide moiety or a branched amino acid, which makes their synthesis a challenge. This protocol provides guidelines for the synthesis of 'head-to-side-chain cyclodepsipeptides' and includes-as an example-a detailed procedure for preparing pipecolidepsin A. Pipecolidepsin was chosen because it is a very complex 'head-to-side-chain cyclodepsipeptide' of marine origin that shows cytotoxicity in several human cancer cell lines. The procedure begins with the synthesis of the noncommercial protected amino acids (2R,3R,4R)-2-{[(9H-fluoren-9-yl)methoxy]carbonylamino}-3-hydroxy-4,5-dimethylhexanoic acid (Fmoc-AHDMHA-OH), Alloc-pipecolic-OH, (4R,5R)-5-((((9H-fluoren-9-yl)methoxy)carbonylamino)-4-oxo-4-(tritylamino)butyl)-2,2-dimethyl-1,3-dioxolane-4-carboxylic acid (Fmoc-DADHOHA(acetonide, Trt))-OH and the pseudodipeptide (2R,3R,4R)-3-hydroxy-2,4,6-trimethylheptanoic acid ((HTMHA)-D-Asp(OtBu)-OH). It details the assembly of the depsipeptidic skeleton using a fully solid-phase approach (typically on an amino polystyrene resin coupled to 3-(4-hydroxymethylphenoxy)propionic acid (AB linker)), including the key ester formation step. It concludes by describing the macrocyclization step performed on solid phase, and the global deprotection and cleavage of the cyclodepsipeptide from the resin using a trifluoroacetic acid-H2O-triisopropylsilane (TFA-H2O-TIS; 95:2.5:2.5) cocktail, as well as the final purification by semipreparative HPLC. The entire procedure takes â¼2 months to complete.
RESUMEN
The last decade has witnessed a renaissance of peptides as drugs. This progress, together with advances in the structural behavior of peptides, has attracted the interest of the pharmaceutical industry in these molecules as potential APIs. In the past, major peptide-based drugs were inspired by sequences extracted from natural structures of low molecular weight. In contrast, nowadays, the peptides being studied by academic and industrial groups comprise more sophisticated sequences. For instance, they consist of long amino acid chains and show a high tendency to form aggregates. Some researchers have claimed that preparing medium-sized proteins is now feasible with chemical ligation techniques, in contrast to medium-sized peptide syntheses. The complexity associated with the synthesis of certain peptides is exemplified by the so-called "difficult peptides", a concept introduced in the 80's. This refers to sequences that show inter- or intra-molecular ß-sheet interactions significant enough to form aggregates during peptide synthesis. These structural associations are stabilized and mediated by non-covalent hydrogen bonds that arise on the backbone of the peptide and-depending on the sequence-are favored. The tendency of peptide chains to aggregate is translated into a list of common behavioral features attributed to "difficult peptides" which hinder their synthesis. In this regard, this manuscript summarizes the strategies used to overcome the inherent difficulties associated with the synthesis of known "difficult peptides". Here we evaluate several external factors, as well as methods to incorporate chemical modifications into sequences, in order to describe the strategies that are effective for the synthesis of "difficult peptides". These approaches have been classified and ordered to provide an extensive guide for achieving the synthesis of peptides with the aforementioned features.
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Péptidos/síntesis química , Enlace de Hidrógeno , Estructura Molecular , Péptidos/químicaRESUMEN
DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines.
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Huella de ADN/métodos , ADN/metabolismo , Depsipéptidos/metabolismo , Sustancias Intercalantes/metabolismo , Algoritmos , ADN/química , ADN/genética , Depsipéptidos/química , Elasticidad , Sustancias Intercalantes/química , Cinética , Ligandos , Modelos Moleculares , Conformación de Ácido Nucleico , Pinzas Ópticas , Unión Proteica , Termodinámica , Factores de TiempoRESUMEN
The 2-methoxy-4-methylsulfinylbenzyl alcohol (Mmsb-OH) safety-catch linker has been described as a useful tool to overcome two obstacles in peptide synthesis: the solubility and fragment condensation of peptides. The incorporation of the linker into an insoluble peptide target, thereby allowing the conjugation of a poly-Lys as a "solubilizing tag", notably enhanced the solubility of the peptide. The selective conditions that remove that linker favored its incorporation as a semipermanent C-terminal protecting group, thereby allowing fragment condensation of peptides.
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Alcoholes Bencílicos/química , Lisina/química , Fragmentos de Péptidos/síntesis química , Péptidos/síntesis química , Secuencia de Aminoácidos , Ácidos Carboxílicos , Estructura Molecular , Fragmentos de Péptidos/química , Péptidos/químicaRESUMEN
The synthesis of a new small library of quinoxaline-containing peptides is described. After cytotoxic evaluation in four human cancer cell lines, as well as detailed biological studies, it was found that the most active compound, RZ2, promotes the formation of acidic compartments, where it accumulates, blocking the progression of autophagy. Further disruption of the mitochondrial membrane potential and an increase in mitochondrial ROS was observed, causing cells to undergo apoptosis. Given its cytotoxic activity and protease-resistant features, RZ2 could be a potential drug candidate for cancer treatment and provide a basis for future research into the crosstalk between autophagy and apoptosis and its relevance in cancer therapy.
RESUMEN
The use of 2-methoxy-4-methylsulfinylbenzyl (Mmsb) as a new backbone amide-protecting group that acts as a safety-catch structure is proposed. Mmsb, which is stable during the elongation of the sequence and trifluoroacetic acid-mediated cleavage from the resin, improves the synthetic process as well as the properties of the quasi-unprotected peptide. Mmsb offers the possibility of purifying and characterizing complex peptide sequences, and renders the target peptide after NH4 I/TFA treatment and subsequent ether precipitation to remove the cleaved Mmsb moiety. First, the "difficult peptide" sequence H-(Ala)10-NH2 was selected as a model to optimize the new protecting group strategy. Second, the complex, bioactive Ac-(RADA)4-NH2 sequence was chosen to validate this methodology. The improvements in solid-phase peptide synthesis combined with the enhanced solubility of the quasi-unprotected peptides, as compared with standard sequences, made it possible to obtain purified Ac-(RADA)4-NH2. To extend the scope of the approach, the challenging Aß(1-42) peptide was synthesized and purified in a similar manner. The proposed Mmsb strategy opens up the possibility of synthesizing other challenging small proteins.
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Compuestos de Bencilo/química , Péptidos/síntesis química , Técnicas de Síntesis en Fase Sólida/métodos , Sulfuros/química , Amidas/química , Secuencia de Aminoácidos , Péptidos beta-Amiloides/síntesis química , Péptidos beta-Amiloides/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Péptidos/química , Ácido Trifluoroacético/químicaRESUMEN
The design and synthesis of Lys- and Arg-containing peptides as solubilizing tags were studied to evaluate their influence on polarity. The relevance of spatial arrangement of polar groups, in α- or ε-amino positions, was confirmed by chromatographic analysis of a rational PolyLys-based synthesized structure. The most promising of the solubilizing tags here analyzed was conjugated to a commercial water-insoluble drug (indomethacin) as proof of concept.
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Antiinflamatorios no Esteroideos/química , Indometacina/química , Péptidos/química , Polilisina/química , Estructura Molecular , SolubilidadRESUMEN
In the search for new drug candidates for DNA recognition, affinity and sequence selectivity are two of the most important features. NMe-azathiocoraline, a synthetic antitumor bisintercalator derived from the natural marine product thiocoraline, shows similar potency to the parent compound, as well as possessing enhanced stability. Analysis of the DNA-binding selectivity of NMe-azathiocoraline by DNase I footprinting using universal substrates with all 136 tetranucleotides and all possible symmetrical hexanucleotide sequences revealed that, although this ligand binds to all CpG steps with lower affinities than thiocoraline, it displays additional binding to AT-rich sites. Moreover, fluorescence melting studies showed a strong interaction of the synthetic molecule with CACGTG and weaker binding to ACATGT and AGATCT. These findings demonstrate that NMe-azathiocoraline has the same mode of action as thiocoraline, mimicking its DNA-binding selectivity despite the substitution of its thioester bonds by NMe-amide bridges.
RESUMEN
The marine environment is a rich source of metabolites with potential therapeutic properties and applications for humans. Here we describe the first isolation, solid-phase total synthesis, and full structural assignment of a new class of cyclodepsipeptides from the Madagascan sponge Ecionemia acervus that shows in vitro cytotoxic activities at submicromolar concentrations. Seven structures belonging to a new family of compounds, given the general name stellatolides, were characterized. The sequence and stereochemistry of all the amino acids in these molecules were established by a combination of spectroscopic analysis, chemical degradation, and derivatization studies. Furthermore, the complete structure of stellatolide A was confirmed by an efficient solid-phase method for the first total synthesis and the full structural assignment of this molecule, including the asymmetric synthesis of the unique ß-hydroxy acid moiety (Z)-3-hydroxy-6,8-dimethylnon-4-enoic acid.
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Depsipéptidos/aislamiento & purificación , Poríferos/química , Animales , Depsipéptidos/síntesis química , Depsipéptidos/química , Estructura Molecular , Espectrometría de Masa por Ionización de ElectrosprayAsunto(s)
Disulfuros/química , Péptidos/química , Péptidos/uso terapéutico , Animales , Humanos , Péptidos/farmacologíaRESUMEN
Phakellistatins is one of the families of Pro-rich cyclic peptides whose synthetic counterparts have revealed cytotoxicities that differ greatly from those displayed by their corresponding natural ones. This is also the case of the last member isolated from this family, phakellistatin19, an octacyclopeptide containing three Pro moieties and a high percentage of apolar residues. Exhaustive NMR studies on the synthetic and natural phakellistatin 19 have been performed in order to find a plausible explanation for this intriguing behavior. Moreover, taking advantage of phakellistatin's framework, analogues with different cis/trans geometry at the key prolyl peptide bonds were designed, covering a promising conformational space that could not be reached by the natural peptide. By introduction of proline surrogates (Ψ(Me,Me)pro residues) in phakellistatin 19, which effectively increases the percentage of cis conformation in the final peptides, this translates into enhanced biological activity, therefore "rescuing" an otherwise inactive cyclopeptide.
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Péptidos Cíclicos/química , Línea Celular Tumoral , Humanos , Conformación Molecular , Resonancia Magnética Nuclear Biomolecular , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Prolina/análogos & derivados , Prolina/química , EstereoisomerismoRESUMEN
Pipecolidepsin A is a head-to-side-chain cyclodepsipeptide isolated from the marine sponge Homophymia lamellosa. This compound shows relevant cytotoxic activity in three human tumour cell lines and has unique structural features, with an abundance of non-proteinogenic residues, including several intriguing amino acids. Although the moieties present in the structure show high synthetic difficulty, the cornerstone is constituted by the unprecedented and highly hindered γ-branched ß-hydroxy-α-amino acid D-allo-(2R,3R,4R)-2-amino-3-hydroxy-4,5-dimethylhexanoic acid (AHDMHA) residue, placed at the branching ester position and surrounded by the four demanding residues L-(2S,3S,4R)-3,4-dimethylglutamine, (2R,3R,4S)-4,7-diamino-2,3-dihydroxy-7-oxoheptanoic acid, D-allo-Thr and L-pipecolic acid. Here we describe the first total synthesis of a D-allo-AHDMHA-containing peptide, pipecolidepsin A, thus allowing chemical structure validation of the natural product and providing a robust synthetic strategy to access other members of the relevant head-to-side-chain family in a straightforward manner.
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Depsipéptidos/farmacología , Neoplasias/tratamiento farmacológico , Ácidos Pipecólicos/farmacología , Animales , Línea Celular Tumoral , Depsipéptidos/síntesis química , Depsipéptidos/química , Femenino , Células HT29 , Células Hep G2 , Humanos , Células MCF-7 , Masculino , Ácidos Pipecólicos/síntesis química , Ácidos Pipecólicos/química , Poríferos/metabolismo , Relación Estructura-ActividadRESUMEN
The natural compounds triostin and thiocoraline are potent antitumor agents that act as DNA bisintercalators. From a pharmaceutical point of view, these compounds are highly attractive although they present a low pharmacokinetic profile, in part due to their low solubility. Synthetically, they represent a tour de force because no robust strategies have been developed to access a broad range of these bicyclic (depsi)peptides in a straightforward manner. Here we describe solid-phase strategies to synthesize new bisintercalators, such as thiocoraline-triostin hybrids, as well as analogues bearing soluble tags. Orthogonal protection schemes (up to five from: Fmoc, Boc Alloc, pNZ, o-NBS, and Troc), together with the right concourse of the coupling reagents (HOSu, HOBt, HOAt, Oxyma, EDC, DIPCDI, PyAOP, PyBOP, HATU, COMU), were crucial to establish the synthetic plan. In vitro studies and structure-activity relationships have been shown trends in the structure-activity relationship that will facilitate the design of new bisintercalators.
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Antineoplásicos/síntesis química , Química Farmacéutica/métodos , Depsipéptidos/química , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Supervivencia Celular , Células HT29 , Humanos , Modelos Químicos , Estructura Molecular , Quinoxalinas/química , Relación Estructura-ActividadRESUMEN
Since the late 1980s, a large number of depsipeptides that contain a new topography, referred to as "head-to-side-chain" cyclodepsipeptides, have been isolated and characterized. These peptides present a unique structural arrangement that comprises a macrocyclic region closed through an ester bond between the C-terminus and a ß-hydroxyl group, and terminated with a polyketide moiety or a more simple branched aliphatic acid. This structural pattern, the presence of unique and complex residues, and relevant bioactivity are the main features shared by all the members of this new class of depsipeptides, which are reviewed herein.
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Organismos Acuáticos/química , Depsipéptidos/aislamiento & purificación , Animales , Depsipéptidos/química , Depsipéptidos/farmacología , Ácidos Grasos/química , Ácidos Grasos/aislamiento & purificación , Ácidos Grasos/farmacología , Humanos , Policétidos/química , Policétidos/aislamiento & purificación , Policétidos/farmacologíaRESUMEN
Cys-disulfide bonds contribute to the stabilization of peptide and protein structures. The synthesis of these molecules requires a proper protection of Cys residues, which is crucial to prevent side-reactions and also to achieve the correct Cys connectivity. Here we undertook a mechanistic study of a set of well-known acid-labile Cys protecting groups, as well other new promising groups, in order to better understand the nature of their acid-lability. The stability of the carbocation generated during the acid treatment was found to have a direct impact on the removal of the protective groups from the corresponding protected Cys-containing peptides. Hence a combination of steric and conjugative effects determines the stability of the carbocations generated. Here we propose diphenylmethyl (Dpm) as a promising protecting group on the basis of its intermediate relative carbocation stability. All the optimized geometries and energies presented in this study were determined using a B3LYP/6-31G(d,p) calculation. The results discussed herein may be of broader applicability for the development of new protecting groups.