RESUMEN
In addition to a variety of viral-glycoprotein receptors (e.g., heparan sulfate, Niemann-Pick C1, etc.), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), from the C-type lectin receptor family, plays one of the most important pathogenic functions for a wide range of viruses (e.g., Ebola, human cytomegalovirus (HCMV), HIV-1, severe acute respiratory syndrome coronavirus 2, etc.) that invade host cells before replication; thus, its inhibition represents a relevant extracellular antiviral therapy. We report two novel p-tBu-calixarene glycoclusters 1 and 2, bearing tetrahydroxamic acid groups, which exhibit micromolar inhibition of soluble DC-SIGN binding and provide nanomolar IC50 inhibition of both DC-SIGN-dependent Jurkat cis-cell infection by viral particle pseudotyped with Ebola virus glycoprotein and the HCMV-gB-recombinant glycoprotein interaction with monocyte-derived dendritic cells expressing DC-SIGN. A unique cooperative involvement of sugar, linker, and calixarene core is likely behind the strong avidity of DC-SIGN for these low-valent systems. We claim herein new promising candidates for the rational development of a large spectrum of antiviral therapeutics.
Asunto(s)
Calixarenos/química , Moléculas de Adhesión Celular/antagonistas & inhibidores , Glicoconjugados/metabolismo , Glicoproteínas/antagonistas & inhibidores , Ácidos Hidroxámicos/química , Lectinas Tipo C/antagonistas & inhibidores , Fenoles/química , Receptores de Superficie Celular/antagonistas & inhibidores , Proteínas Virales/antagonistas & inhibidores , Antivirales/química , Antivirales/metabolismo , Antivirales/farmacología , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Citomegalovirus/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Ebolavirus/fisiología , Glicoconjugados/química , Glicoconjugados/farmacología , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Células Jurkat , Lectinas Tipo C/metabolismo , Modelos Biológicos , Unión Proteica , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure-activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product's biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence á´ -Ala-Dha-Sar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide.
RESUMEN
Biofilm formation is a key mechanism of antimicrobial resistance. We have recently reported two classes of orally bioavailable C-glycosidic inhibitors of the Pseudomonas aeruginosa lectin LecB with antibiofilm activity. They proved efficient in target binding, were metabolically stable, nontoxic, selective, and potent in inhibiting formation of bacterial biofilm. Here, we designed and synthesized six new carboxamides and 24 new sulfonamides for a detailed structure-activity relationship for two clinically representative LecB variants. Sulfonamides generally showed higher inhibition compared to carboxamides, which was rationalized based on crystal structure analyses. Substitutions at the thiophenesulfonamide increased binding through extensive contacts with a lipophilic protein patch. These metabolically stable compounds showed a further increase in potency toward the target and in biofilm inhibition assays. In general, we established the structure-activity relationship for these promising antibiofilm agents and showed that modification of the sulfonamide residue bears future optimization potential.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Glicósidos/química , Lectinas/antagonistas & inhibidores , Pseudomonas aeruginosa/fisiología , Animales , Antibacterianos/química , Antibacterianos/metabolismo , Sitios de Unión , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Lectinas/metabolismo , Ratones , Microsomas Hepáticos/metabolismo , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Opportunistic Gram-negative Pseudomonas aeruginosa uses adhesins (e.g., LecA and LecB lectins, type VI pili and flagella) and iron to invade host cells with the formation of a biofilm, a thick barrier that protects bacteria from drugs and host immune system. Hindering iron uptake and disrupting adhesins' function could be a relevant antipseudomonal strategy. To test this hypothesis, we designed an iron-chelating glycocluster incorporating a tetrahydroxamic acid and α-l-fucose bearing linker to interfere with both iron uptake and the glycan recognition process involving the LecB lectin. Iron depletion led to increased production of the siderophore pyoverdine by P. aeruginosa to counteract the loss of iron uptake, and strong biofilm inhibition was observed not only with the α-l-fucocluster (72%), but also with its α-d-manno (84%), and α-d-gluco (92%) counterparts used as negative controls. This unprecedented finding suggests that both LecB and biofilm inhibition are closely related to the presence of hydroxamic acid groups.
Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Glicoconjugados/farmacología , Ácidos Hidroxámicos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Adhesinas Bacterianas/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Glicoconjugados/síntesis química , Glicoconjugados/química , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Pathogenic micro-organisms utilize protein receptors (lectins) in adhesion to host tissues, a process that in some cases relies on the interaction between lectins and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analysed by X-ray crystallography the structures of the LecB lectin from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 4% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we propose a general strategy which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.
Asunto(s)
Lectinas/metabolismo , Oligosacáridos/metabolismo , Sitios de Unión , Calcio/química , Calcio/metabolismo , Cristalografía por Rayos X , Glucosamina/química , Glucosamina/metabolismo , Lectinas/química , Lectinas/genética , Ligandos , Conformación Molecular , Simulación de Dinámica Molecular , Mutación , Oligosacáridos/química , Unión Proteica , Pseudomonas aeruginosa/químicaRESUMEN
Innate immunity is the first line of defense against pathogens and predators. To initiate a response, it relies on the detection of invaders, where lectin-carbohydrate interactions play a major role. O-Methylated glycans were previously identified as non-self epitopes and conserved targets for defense effector proteins belonging to the tectonin superfamily. Here, we present two crystal structures of Tectonin 2 from the mushroom Laccaria bicolor in complex with methylated ligands, unraveling the molecular basis for this original specificity. Furthermore, they revealed the formation of a ball-shaped tetramer with 24 binding sites distributed at its surface, resembling a small virus capsid. Based on the crystal structures, a methylation recognition motif was identified and found in the sequence of many tectonins from bacteria to human. Our results support a key role of tectonins in innate defense based on a distinctive and conserved type of lectin-glycan interaction.
Asunto(s)
Laccaria/inmunología , Lectinas/química , Lectinas/metabolismo , Polisacáridos/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Inmunidad Innata , Laccaria/química , Laccaria/metabolismo , Metilación , Modelos Moleculares , Polisacáridos/química , Multimerización de Proteína , Estructura Terciaria de Proteína , Dispersión del Ángulo PequeñoRESUMEN
The opportunistic Gram-negative bacterium Pseudomonas aeruginosa is a leading pathogen for infections of immuno-compromised patients and those suffering from cystic fibrosis. Its ability to switch from planktonic life to aggregates, forming the so-called biofilms, is a front-line mechanism of antimicrobial resistance. The bacterial carbohydrate-binding protein LecB is an integral component and necessary for biofilm formation. Here, we report a new class of drug-like low molecular weight inhibitors of the lectin LecB with nanomolar affinities and excellent receptor binding kinetics and thermodynamics. This class of glycomimetic inhibitors efficiently blocked biofilm formation of P. aeruginosa in vitro while the natural monovalent carbohydrate ligands failed. Furthermore, excellent selectivity and pharmacokinetic properties were achieved. Notably, two compounds showed good oral bioavailability, and high compound concentrations in plasma and urine were achieved in vivo.
Asunto(s)
Biopelículas/efectos de los fármacos , Cinamatos/farmacología , Lectinas/antagonistas & inhibidores , Pseudomonas aeruginosa/efectos de los fármacos , Sulfonamidas/farmacología , Administración Oral , Disponibilidad Biológica , Cinamatos/administración & dosificación , Cinamatos/química , Relación Dosis-Respuesta a Droga , Cinética , Lectinas/metabolismo , Conformación Molecular , Pseudomonas aeruginosa/metabolismo , Relación Estructura-Actividad , Sulfonamidas/administración & dosificación , Sulfonamidas/química , TermodinámicaRESUMEN
Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing "pathoblockers" preventing biofilm formation and virulence. A covalent lectin inhibitor specific to a carbohydrate binding site is described for the first time. Its application in the LecA-specific inâ vitro imaging of biofilms formed by P. aeruginosa is also reported.
Asunto(s)
Lectinas/metabolismo , Pseudomonas aeruginosa/fisiología , Adhesinas Bacterianas/química , Adhesinas Bacterianas/metabolismo , Sitios de Unión , Biopelículas/efectos de los fármacos , Biopelículas/crecimiento & desarrollo , Carbohidratos/química , Cristalografía por Rayos X , Diseño de Fármacos , Compuestos Epoxi/química , Compuestos Epoxi/metabolismo , Compuestos Epoxi/farmacología , Lectinas/antagonistas & inhibidores , Factores de Virulencia/antagonistas & inhibidores , Factores de Virulencia/metabolismoRESUMEN
Infections with the Gram-negative bacterium Pseudomonas aeruginosa result in a high mortality among immunocompromised patients and those with cystic fibrosis. The pathogen can switch from planktonic life to biofilms, and thereby shields itself against antibiotic treatment and host immune defense to establish chronic infections. The bacterial protein LecA, a C-type lectin, is a virulence factor and an integral component for biofilm formation. Inhibition of LecA with its carbohydrate ligands results in reduced biofilm mass, a potential Achilles heel for treatment. Here, we report the development and optimization of a fluorescence polarization-based competitive binding assay with LecA for application in screening of potential inhibitors. As a consequence of the low affinity of d-galactose for LecA, the fluorescent ligand was optimized to reduce protein consumption in the assay. The assay was validated using a set of known inhibitors of LecA and IC50 values in good agreement with the known Kd values were obtained. Finally, we employed the optimized assay to screen sets of synthetic thio-galactosides and natural blood group antigens and report their structure-activity relationship. In addition, we evaluated a multivalent fluorescent assay probe for LecA and report its applicability in an inhibition assay.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Colorantes Fluorescentes/farmacología , Galactosa/farmacología , Pseudomonas aeruginosa/química , Adhesinas Bacterianas/química , Unión Competitiva/efectos de los fármacos , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Polarización de Fluorescencia , Colorantes Fluorescentes/química , Galactosa/química , Humanos , Ligandos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Infections with Pseudomonas aeruginosa have become a concerning threat in hospital-acquired infections and for cystic fibrosis patients. The major problem leading to high mortality lies in the appearance of drug-resistant strains. Therefore, a vast number of approaches to develop novel anti-infectives is currently pursued. These diverse strategies span from killing (new antibiotics) to disarming (antivirulence) the pathogen. Particular emphasis lies on the development of compounds that inhibit biofilms formed in chronic infections to restore susceptibility toward antibiotics. Numerous promising results are summarized in this perspective. Antibiotics with a novel mode of action will be needed to avoid cross resistance against currently used therapeutic agents. Importantly, antivirulence drugs are expected to yield a significantly reduced rate of resistance development. Most developments are still far from the application. It can however be expected that combination therapies, also containing antivirulence agents, will pave the way toward novel treatment options against P. aeruginosa.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Descubrimiento de Drogas/métodos , Terapia Molecular Dirigida/métodos , Infecciones por Pseudomonas/tratamiento farmacológico , Pseudomonas aeruginosa/efectos de los fármacos , Animales , Antibacterianos/uso terapéutico , Biopelículas/efectos de los fármacos , Humanos , Modelos Moleculares , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/efectos de los fármacosRESUMEN
Selenoglycosides are used as reactive glycosyl donors in the syntheses of oligosaccharides. In addition, such heavy atom analogs of natural glycosides are useful tools for structure determination of their lectin receptors using X-ray crystallography. Some lectins, e.g., members of the tectonin family, only bind to carbohydrate epitopes with O-alkylated ring hydroxy groups. In this context, we report the first synthesis of an O-methylated selenoglycoside, specifically methyl 2-O-methyl-L-selenofucopyranoside, a ligand of the lectin tectonin-2 from the mushroom Laccaria bicolor. The synthetic route required a strategic revision and further optimization due to the intrinsic lability of alkyl selenoglycosides, in particular for the labile fucose. Here, we describe a successful synthetic access to methyl 2-O-methyl-L-selenofucopyranoside in 9 linear steps and 26% overall yield starting from allyl L-fucopyranoside.
RESUMEN
P. aeruginosa causes a substantial number of nosocomial infections and is the leading cause of death of cystic fibrosis patients. This Gram-negative bacterium is highly resistant against antibiotics and further protects itself by forming a biofilm. Moreover, a high genomic variability among clinical isolates complicates therapy. Its lectin LecB is a virulence factor and necessary for adhesion and biofilm formation. We analyzed the sequence of LecB variants in a library of clinical isolates and demonstrate that it can serve as a marker for strain family classification. LecB from the highly virulent model strain PA14 presents 13% sequence divergence with LecB from the well characterized PAO1 strain. These differences might result in differing ligand binding specificities and ultimately in reduced efficacy of drugs directed towards LecB. Despite several amino acid variations at the carbohydrate binding site, glycan array analysis showed a comparable binding pattern for both variants. A common high affinity ligand could be identified and after its chemoenzymatic synthesis verified in a competitive binding assay: an N-glycan presenting two blood group O epitopes (H-type 2 antigen). Molecular modeling of the complex suggests a bivalent interaction of the ligand with the LecB tetramer by bridging two separate binding sites. This binding rationalizes the strong avidity (35 nM) of LecBPA14 to this human fucosylated N-glycan. Biochemical evaluation of a panel of glycan ligands revealed that LecBPA14 demonstrated higher glycan affinity compared to LecBPAO1 including the extraordinarily potent affinity of 70 nM towards the monovalent human antigen Lewisa. The structural basis of this unusual high affinity ligand binding for lectins was rationalized by solving the protein crystal structures of LecBPA14 with several glycans.
RESUMEN
Biofilm formation and chronic infections with Pseudomonas aeruginosa depend on lectins produced by the bacterium. The bacterial C-type lectin LecB binds to the two monosaccharides l-fucose and d-mannose and conjugates thereof. Previously, d-mannose derivatives with amide and sulfonamide substituents at C6 were reported as potent inhibitors of the bacterial lectin LecB and LecB-mediated bacterial surface adhesion. Because d-mannose establishes a hydrogen bond via its 6-OH group with Ser23 of LecB in the crystal structure and may be beneficial for binding affinity, we extended d-mannose and synthesized mannoheptoses bearing the free 6-OH group as well as amido and sulfonamido-substituents at C7. Two series of diastereomeric mannoheptoses were synthesized and the stereochemistry was determined by X-ray crystallography. The potency of the mannoheptoses as LecB inhibitors was assessed in a competitive binding assay. The data reveal a diastereoselectivity of LecB for (6S)-mannoheptose derivatives with increased activity over methyl α-d-mannoside.
Asunto(s)
Heptosas/síntesis química , Lectinas/antagonistas & inhibidores , Pseudomonas aeruginosa/química , Aminas/química , Cristalografía por Rayos X , Heptosas/química , Ligandos , Metilmanósidos/síntesis química , Metilmanósidos/química , Nitrilos/síntesis química , Nitrilos/química , Unión Proteica , Pseudomonas aeruginosa/patogenicidadRESUMEN
Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen with high antibiotic resistance. Its lectin LecB was identified as a virulence factor and is relevant in bacterial adhesion and biofilm formation. Inhibition of LecB with carbohydrate-based ligands results in a decrease in toxicity and biofilm formation. We recently discovered two classes of potent drug-like glycomimetic inhibitors, that is, sulfonamides and cinnamides of d-mannose. Here, we describe the chemical synthesis and biochemical evaluation of more than 20â derivatives with increased potency compared to the unsubstituted cinnamide. The structure-activity relationship (SAR) obtained and the extended biophysical characterization allowed the experimental determination of the binding mode of these cinnamides with LecB. The established surface binding mode now allows future rational structure-based drug design. Importantly, all glycomimetics tested showed extended receptor residence times with half-lives in the 5-20â min range, a prerequisite for therapeutic application. Thus, the glycomimetics described here provide an excellent basis for future development of anti-infectives against this multidrug-resistant pathogen.
RESUMEN
The rise of resistances against antibiotics in bacteria is a major threat for public health and demands the development of novel antibacterial therapies. Infections with Pseudomonas aeruginosa are a severe problem for hospitalized patients and for patients suffering from cystic fibrosis. These bacteria can form biofilms and thereby increase their resistance towards antibiotics. The bacterial lectin LecB was shown to be necessary for biofilm formation and the inhibition with its carbohydrate ligands resulted in reduced amounts of biofilm. The natural ligands for LecB are glycosides of D-mannose and L-fucose, the latter displaying an unusual strong affinity. Interestingly, although mannosides are much weaker ligands for LecB, they do form an additional hydrogen bond with the protein in the crystal structure. To analyze the individual contributions of the methyl group in fucosides and the hydroxymethyl group in mannosides to the binding, we designed and synthesized derivatives of these saccharides. We report glycomimetic inhibitors that dissect the individual interactions of their saccharide precursors with LecB and give insight into the biophysics of binding by LecB. Furthermore, theoretical calculations supported by experimental thermodynamic data suggest a perturbed hydrogen bonding network for mannose derivatives as molecular basis for the selectivity of LecB for fucosides. Knowledge gained on the mode of interaction of LecB with its ligands at ambient conditions will be useful for future drug design.
Asunto(s)
Carbohidratos/química , Lectinas/química , Monosacáridos/química , Pseudomonas aeruginosa/química , Unión Competitiva , Biopelículas , Biofisica , Calorimetría , Cromatografía Liquida , Resonancia Magnética Nuclear BiomolecularRESUMEN
Hospital-acquired bacterial infections, especially with Gram-negative pathogens, present a major threat due to the rapid spread of antibiotic-resistant strains. Targeting mechanisms of bacterial virulence has recently appeared as a promising new therapeutic paradigm. Biofilm formation is a bacterial lifestyle, which offers a survival advantage through its protective matrix against host immune defense and antibiotic treatment. Interfering with biogenesis of adhesive organelles, bacterial communication or carbohydrate-mediated adhesion as anti-biofilm strategies are reviewed.