RESUMEN
A ring-closing metathesis (RCM) - peptide coupling - ruthenium-catalyzed azide alkyne cycloaddition (RuAAC) strategy was developed to synthesize a tricyclic hexapeptide in which the side chain to side chain connectivity pattern resulted in a mimic with a topology that effectively mimics the bioactivity of vancomycin as a potent binder of the bacterial cell wall D-Ala-D-Ala dipeptide sequence and more importantly being an effective inhibitor of bacterial growth.
Asunto(s)
Rutenio , Catálisis , Ciclización , Dipéptidos , Rutenio/química , Vancomicina/química , Vancomicina/farmacologíaRESUMEN
The continued rise of antibiotic resistance is a global concern that threatens to undermine many aspects of modern medical practice. Key to addressing this threat is the discovery and development of new antibiotics that operate by unexploited modes of action. The so-called calcium-dependent lipopeptide antibiotics (CDAs) are an important emerging class of natural products that provides a source of new antibiotic agents rich in structural and mechanistic diversity. Notable in this regard is the subset of CDAs comprising the laspartomycins and amphomycins/friulimicins that specifically target the bacterial cell wall precursor undecaprenyl phosphate (C55-P). In this study we describe the design and synthesis of new C55-P-targeting CDAs with structural features drawn from both the laspartomycin and amphomycin/friulimicin classes. Assessment of these lipopeptides revealed previously unknown and surprisingly subtle structural features that are required for antibacterial activity. High-resolution crystal structures further indicate that the amphomycin/friulimicin-like lipopeptides adopt a unique crystal packing that governs their interaction with C55-P and provides an explanation for their antibacterial effect. In addition, live-cell microscopy studies provide further insights into the biological activity of the C55-P targeting CDAs highlighting their unique mechanism of action relative to the clinically used CDA daptomycin.
RESUMEN
For canonical asparagine glycosylation, the primary amino acid sequence that directs glycosylation at specific asparagine residues is well-established. Here we reveal that a recently discovered bacterial enzyme EarP, that transfers rhamnose to a specific arginine residue in its acceptor protein EF-P, specifically recognizes a ß-hairpin loop. Notably, while the in vitro rhamnosyltransferase activity of EarP is abolished when presented with linear substrate peptide sequences derived from EF-P, the enzyme readily glycosylates the same sequence in a cyclized ß-hairpin mimic. Additional studies with other substrate-mimicking cyclic peptides revealed that EarP activity is sensitive to the method used to induce cyclization and in some cases is tolerant to amino acid sequence variation. Using detailed NMR approaches, we established that the active peptide substrates all share some degree of ß-hairpin formation, and therefore conclude that the ß-hairpin epitope is the major determinant of arginine-rhamnosylation by EarP. Our findings add a novel recognition motif to the existing knowledge on substrate specificity of protein glycosylation, and are expected to guide future identifications of rhamnosylation sites in other protein substrates.
RESUMEN
There is a constant need for new therapeutic compounds. Fungi have proven to be an excellent, but underexplored source for biologically active compounds with therapeutic potential. Here, we combine mycology, embryology and chemistry by testing secondary metabolites from more than 10,000 species of fungi for biological activity using developing zebrafish (Danio rerio) embryos. Zebrafish development is an excellent model for high-throughput screening. Development is rapid, multiple cell types are assessed simultaneously and embryos are available in high numbers. We found that 1,526 fungal strains produced secondary metabolites with biological activity in the zebrafish bioassay. The active compounds from 39 selected fungi were purified by liquid-liquid extraction and preparative HPLC. 34 compounds were identified by a combination of chemical analyses, including LCMS, UV-Vis spectroscopy/ spectrophotometry, high resolution mass spectrometry and NMR. Our results demonstrate that fungi express a wide variety of biologically active compounds, consisting of both known therapeutic compounds as well as relatively unexplored compounds. Understanding their biological activity in zebrafish may provide insight into underlying biological processes as well as mode of action. Together, this information may provide the first step towards lead compound development for therapeutic drug development.
Asunto(s)
Hongos/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Descubrimiento de Drogas/métodos , Desarrollo Embrionario/efectos de los fármacos , Hongos/química , Ensayos Analíticos de Alto Rendimiento/métodos , Espectroscopía de Fotoelectrones , Pez Cebra/embriologíaRESUMEN
Laspartomycin C is a lipopeptide antibiotic with activity against a range of Gram-positive bacteria including drug-resistant pathogens. We report the first total synthesis of laspartomycin C as well as a series of structural variants. Laspartomycin C was found to specifically bind undecaprenyl phosphate (C55-P) and inhibit formation of the bacterial cell wall precursor lipid II. While several clinically used antibiotics target the lipid II pathway, there are no approved drugs that act on its C55-P precursor.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Pared Celular/efectos de los fármacos , Lipopéptidos/síntesis química , Lipopéptidos/farmacología , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Staphylococcus aureus/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Uridina Difosfato Ácido N-Acetilmurámico/análogos & derivados , Uridina Difosfato Ácido N-Acetilmurámico/antagonistas & inhibidoresRESUMEN
The bacterial adhesion lectin LecA is an attractive target for interference with the infectivity of its producer P.â aeruginosa. Divalent ligands with two terminal galactoside moieties connected by an alternating glucose-triazole spacer were previously shown to be very potent inhibitors. In this study, we chose to prepare a series of derivatives with various new substituents in the spacer in hopes of further enhancing the LecA inhibitory potency of the molecules. Based on the binding mode, modifications were made to the spacer to enable additional spacer-protein interactions. The introduction of positively charged, negatively charged, and also lipophilic functional groups was successful. The compounds were good LecA ligands, but no improved binding was seen, even though altered thermodynamic parameters were observed by isothermal titration calorimetry (ITC).
RESUMEN
A new divalent highly potent inhibitor of the Pseudomonas aeruginosa lectin and virulence factor LecA was prepared. It contains two thiourea linkages which were found to be in the Z,Z isomeric form. This brings the spacer into an elongated conformation required to bridge the two binding sites, which results in the chelating binding mode responsible for the high potency.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Antibacterianos/química , Antibacterianos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Tiourea/análogos & derivados , Tiourea/farmacología , Factores de Virulencia/metabolismo , Humanos , Lectinas/antagonistas & inhibidores , Lectinas/metabolismo , Simulación del Acoplamiento Molecular , Infecciones por Pseudomonas/tratamiento farmacológico , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Factores de Virulencia/antagonistas & inhibidoresRESUMEN
Multivalent galactosides inhibiting Pseudomonas aeruginosa biofilms may help control this problematic pathogen. To understand the binding mode of tetravalent glycopeptide dendrimer GalAG2 [(Gal-ß-OC6H4CO-Lys-Pro-Leu)4(Lys-Phe-Lys-Ile)2Lys-His-Ile-NH2] to its target lectin LecA, crystal structures of LecA complexes with divalent analog GalAG1 [(Gal-ß-OC6H4CO-Lys-Pro-Leu)2Lys-Phe-Lys-Ile-NH2] and related glucose-triazole linked bis-galactosides 3u3 [Gal-ß-O(CH2)n-(C2HN3)-4-Glc-ß-(C2HN3)-[ß-Glc-4-(N3HC2)]2-(CH2)n-O-ß-Gal (n = 1)] and 5u3 (n = 3) were obtained, revealing a chelate bound 3u3, cross-linked 5u3, and monovalently bound GalAG1. Nevertheless, a chelate bound model better explaining their strong LecA binding and the absence of lectin aggregation was obtained by modeling for all three ligands. A model of the chelate bound GalAG2·LecA complex was also obtained rationalizing its unusually tight LecA binding (KD = 2.5 nM) and aggregation by lectin cross-linking. The very weak biofilm inhibition with divalent LecA inhibitors suggests that lectin aggregation is necessary for biofilm inhibition by GalAG2, pointing to multivalent glycoclusters as a unique opportunity to control P. aeruginosa biofilms.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Galactósidos/metabolismo , Pseudomonas aeruginosa/metabolismo , Adhesinas Bacterianas/química , Sitios de Unión , Secuencia de Carbohidratos , Complejos de Coordinación/química , Cristalografía por Rayos X , Galactósidos/química , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia MolecularRESUMEN
The calcium-dependent lipopeptide antibiotics represent a promising new class of antimicrobials for use in combating drug-resistant bacteria. At present, daptomycin is the only such lipopeptide used clinically and displays potent antimicrobial activity against a number of pathogenic Gram-positive bacteria. Given the increasing need for new antibiotics, practical synthetic access to unnatural analogues of daptomycin and related antimicrobial lipopeptides is of value. We here report an efficient synthetic route combining solid- and solution-phase techniques that allows for the rapid preparation of daptomycin analogues. Using this approach, four such analogues, including two enantiomeric variants, were synthesized and their antimicrobial activities and hydrolytic stabilities evaluated.
Asunto(s)
Antibacterianos/farmacología , Calcio/química , Daptomicina/farmacología , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Daptomicina/análogos & derivados , Daptomicina/química , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Soluciones , Relación Estructura-ActividadRESUMEN
Herein, we describe the synthesis, structural characterization, and synthetic use as an advanced intermediate of a cross-stapled alkene-bridged hexapeptide to mimic the DE-ring of the lantibiotic nisin. The linear precursor was cyclized by ring-closing metathesis to give the correctly folded bicyclic hexapeptide in a single step, and the four individual diastereoisomers were isolated, structurally assigned and characterized by HPLC, NMR and MS, respectively. The bicyclic hexapeptide was used as a versatile advanced synthon and was modified at its C- and N-terminus, among others, with an azide moiety to access a building block suitable for Cu(I)-catalyzed alkyne-azide cycloaddition-based ligation reactions.
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Alquenos/química , Antibacterianos/química , Antibacterianos/síntesis química , Nisina/química , Nisina/síntesis química , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Relación Dosis-Respuesta a Droga , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nisina/farmacología , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Panton-Valentine Leukocidin (PVL) is a staphylococcal bicomponent pore-forming toxin linked to severe invasive infections. Target-cell and species specificity of PVL are poorly understood, and the mechanism of action of this toxin in Staphylococcus aureus virulence is controversial. Here, we identify the human complement receptors C5aR and C5L2 as host targets of PVL, mediating both toxin binding and cytotoxicity. Expression and interspecies variations of the C5aR determine cell and species specificity of PVL. The C5aR binding PVL component, LukS-PV, is a potent inhibitor of C5a-induced immune cell activation. These findings provide insight into leukocidin function and staphylococcal virulence and offer directions for future investigations into individual susceptibility to severe staphylococcal disease.
Asunto(s)
Toxinas Bacterianas/metabolismo , Exotoxinas/metabolismo , Interacciones Huésped-Patógeno , Leucocidinas/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidad , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidad , Células Cultivadas , Exotoxinas/toxicidad , Humanos , Leucocidinas/toxicidad , Receptores de Quimiocina/metabolismoRESUMEN
Herein we report the Ru(II)-catalyzed double-macrocyclization of a hexapeptide to obtain a mimic of the bicyclic CDE-ring of vancomycin, followed by measurement of its binding affinity for small peptide ligands using ITC.
Asunto(s)
Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Organometálicos/química , Triazoles/síntesis química , Vancomicina/síntesis química , Alquinos/química , Azidas/química , Compuestos Bicíclicos con Puentes/química , Ciclización , Modelos Moleculares , Estructura Molecular , Rutenio/química , Triazoles/química , Vancomicina/análogos & derivados , Vancomicina/químicaRESUMEN
A new class of potent proteasome inhibitors is described, of which the members contain an amino acid inspired sulfonyl fluoride as the electrophilic trap. In total, 24 peptido sulfonyl fluoride inhibitors have been designed and synthesized, which were inspired by the backbone sequences of the proteasome inhibitors bortezomib, epoxomicin, and Cbz-Leu(3)-aldehyde. Nine of them were very potent proteasome inhibitors, the best of which had an IC(50) of 7 nM. A number of the peptido sulfonyl fluoride inhibitors were found to be highly selective for the ß5 proteasome subunit.
Asunto(s)
Péptidos/síntesis química , Inhibidores de Proteasoma/síntesis química , Sulfonas/síntesis química , Células HEK293 , Humanos , Péptidos/química , Péptidos/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/química , Inhibidores de Proteasoma/farmacología , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Sulfonas/química , Sulfonas/farmacologíaRESUMEN
The synthesis of cyclic peptides containing a thioester handle using a sulfo-click linker is reported. These cyclic peptides can be coupled to N-terminal cysteine-containing constructs via native chemical ligation. A successful application of a cyclic peptide bearing a thioester handle in native chemical ligation is shown by a high yielding ligation.
Asunto(s)
Cisteína/análogos & derivados , Cisteína/síntesis química , Péptidos Cíclicos/química , Péptidos Cíclicos/síntesis química , Compuestos de Azufre/química , Compuestos de Azufre/síntesis química , Ligadura , Datos de Secuencia MolecularRESUMEN
The echinocandins represent the most recent class of antifungal drugs. Previous structure-activity relationship studies on these lipopeptides have relied mainly upon semisynthetic derivatives due to their complex chemical structures. A successful strategy for the rapid enantioselective synthesis of the branched fatty acid chain of caspofungin and analogues was developed to synthesize several simplified analogues of caspofungin. The specific minimum inhibitory activity of each mimic was determined against a panel of Candida strains. This approach gave access to new fully synthetic derived caspofungin mimics with high and selective antifungal activities against Candida strains. In addition, the data suggested an important role of the hydroxy proline residue in the bioactive conformation of the macrocyclic peptide ring structure.
Asunto(s)
Antifúngicos/farmacología , Candida/efectos de los fármacos , Equinocandinas/farmacología , Prolina/química , Antifúngicos/síntesis química , Antifúngicos/química , Caspofungina , Relación Dosis-Respuesta a Droga , Equinocandinas/síntesis química , Equinocandinas/química , Lipopéptidos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Echinocandins are a novel class of macrocyclic antifungal peptides that act by inhibiting the ß-(1,3)-D-glucan synthase complex, which is not present in mammalian cells. Due to the large number of hydroxyl groups present in these complex macrocyclic lipopeptides, most structure-activity relationship studies have relied upon semisynthetic derivatives. In order to probe the influence of the cyclic peptide backbone on the antifungal activity we developed a successful strategy for the synthesis of novel echinocandins analogues by on-resin ring closing metathesis or disulfide formation. The specific minimum inhibitory activity of each mimic was determined against Candida albicans. Our results indicate that ring size is an important factor for antifungal activity.
Asunto(s)
Antifúngicos/química , Candida albicans/efectos de los fármacos , Equinocandinas/química , Equinocandinas/farmacología , Inhibidores Enzimáticos/química , Antifúngicos/síntesis química , Antifúngicos/farmacología , Candida albicans/enzimología , Equinocandinas/síntesis química , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glucosiltransferasas/antagonistas & inhibidores , Espectroscopía de Resonancia Magnética , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Técnicas de Síntesis en Fase Sólida/métodos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Despite their enormous diversity in biological function and structure, peptides and proteins are endowed with properties that have induced and stimulated the development of peptidomimetics. Clearly, peptides can be considered as the "stem" of a phylogenetic molecular development tree from which branches of oligomeric peptidomimetics such as peptoids, peptidosulfonamides, urea peptidomimetics, as well as ß-peptides have sprouted. It is still a challenge to efficiently synthesize these oligomeric species, and study their structural and biological properties. Combining peptides and peptidomimetics led to the emergence of peptide-peptidomimetic hybrids in which one or more (proteinogenic) amino acid residues have been replaced with these mimetic residues. In scan-like approaches, the influence of these replacements on biological activity can then be studied, to evaluate to what extent a peptide can be transformed into a peptidomimetic structure while maintaining, or even improving, its biological properties. A central issue, especially with the smaller peptides, is the lack of secondary structure. Important approaches to control secondary structure include the introduction of α,α-disubstituted amino acids, or (di)peptidomimetic structures such as the Freidinger lactam. Apart from intra-amino acid constraints, inter-amino acid constraints for formation of a diversity of cyclic peptides have shaped a thick branch. Apart from the classical disulfide bridges, the repertoire has been extended to include sulfide and triazole bridges as well as the single-, double- and even triple-bond replacements, accessible by the extremely versatile ring-closing alkene/alkyne metathesis approaches. The latter approach is now the method of choice for the secondary structure that presents the greatest challenge for structural stabilization: the α-helix.
Asunto(s)
Aminoácidos/química , Péptidos/química , Peptidomiméticos/química , Proteínas/química , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Peptoides/química , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
We present experimental proof that so-called "flowerlike micelles" exist and that they have some distinctly different properties compared to their "starlike" counterparts. Amphiphilic AB diblock and BAB triblock copolymers consisting of poly(ethylene glycol) (PEG) as hydrophilic A block and thermosensitive poly(N-isopropylacrylamide) (pNIPAm) B block(s) were synthesized via atom transfer radical polymerization (ATRP). In aqueous solutions, both block copolymer types form micelles above the cloud point of pNIPAm. Static and dynamic light scattering measurements in combination with NMR relaxation experiments proved the existence of flowerlike micelles based on pNIPAm(16kDa)-PEG(4kDa)-pNIPAm(16kDa) which had a smaller radius and lower mass and aggregation number than starlike micelles based on mPEG(2kDa)-pNIPAm(16kDa). Furthermore, the PEG surface density was much lower for the flowerlike micelles, which we attribute to the looped configuration of the hydrophilic PEG block. (1)H NMR relaxation measurements showed biphasic T(2) relaxation for PEG, indicating rigid PEG segments close to the micelle core and more flexible distal segments. Even the flexible distal segments were shown to have a lower mobility in the flowerlike micelles compared to the starlike micelles, indicating strain due to loop formation. Taken together, it is demonstrated that self-assemblies of BAB triblock copolymers have their hydrophilic block in a looped conformation and thus indeed adopt a flowerlike conformation.
Asunto(s)
Luz , Espectroscopía de Resonancia Magnética/métodos , Micelas , Dispersión de RadiaciónRESUMEN
Structural mimics comprising 1,4- and 1,5-disubstituted triazole-containing cyclic tripeptides with excellent resemblance toward the DE-ring of vancomycin are conveniently accessible using Cu(I)- or Ru(II)-assisted "click" cyclization.