RESUMEN
A focused library of virtual heterobifunctional ligands was generated in silico and a set of ligands with recombined fragments was synthesized and evaluated for binding to Clostridium difficile toxins. The position of the trisaccharide fragment was used as a reference for filtering docked poses during virtual screening to match the trisaccharide ligand in a crystal structure. The peptoid, a diversity fragment probing the protein surface area adjacent to a known binding site, was generated by a multi-component Ugi reaction. Our approach combines modular fragment-based design with in silico screening of synthetically feasible compounds and lays the groundwork for future efforts in development of composite bifunctional ligands for large clostridial toxins.
Asunto(s)
Clostridioides difficile , Simulación por Computador , Bibliotecas de Moléculas Pequeñas/metabolismo , Toxinas Biológicas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Metabolismo de los Hidratos de Carbono , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Toxinas Biológicas/químicaRESUMEN
The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(ß1-4)Glc, Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc, Fuc(α1-2)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Fuc(α1-4)]GlcNAc(ß1-3)Gal(ß1-4)Glc and Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-3)Gal(ß1-4)Glc. However, the binding of the HMOs is uniformly weak, with apparent affinities ≤10(3 )M(-1). The results of molecular docking simulations, taken together with the experimental binding data, suggest that a disaccharide moiety (lactose or lactosamine) represents the core HMO recognition element for both toxin fragments. The results of a Verocytotoxicity neutralization assay reveal that HMOs do not significantly inhibit the cytotoxic effects of TcdA or TcdB. The absence of protection is attributed to the very weak intrinsic affinities that the toxins exhibit towards the HMOs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Clostridioides difficile/química , Enterotoxinas/metabolismo , Leche Humana/química , Oligosacáridos , Fragmentos de Péptidos/metabolismo , Amino Azúcares/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacología , Sitios de Unión , Secuencia de Carbohidratos , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Enterotoxinas/química , Enterotoxinas/farmacología , Humanos , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Oligosacáridos/análisis , Oligosacáridos/química , Oligosacáridos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Unión Proteica , Espectrometría de Masa por Ionización de Electrospray , Células VeroRESUMEN
The tether employed to covalently attach ß-mannan disaccharide glycoconjugates influences the specificity of rabbit antibodies that protect against Candida albicans. Two glycoconjugates containing (1 â 2)-ß-mannan disaccharides linked to chicken serum albumin (CSA) either via a structurally uniform or via a stereodiversified spacer were prepared and evaluated in immunization trials in mice and rabbits. Immunization with conjugate vaccine possessing a structurally diversified linker induced higher IgG titers against Candida albicans cell wall phosphomannan than a conjugate with a structurally uniform linker. These results suggest that affinity maturation and the specific antibody response can be shifted towards recognition of the desired hapten by employing a linker with diversified configuration.
Asunto(s)
Glicoconjugados/química , Glicoconjugados/inmunología , Haptenos/química , Haptenos/inmunología , Animales , Especificidad de Anticuerpos , Candida albicans/inmunología , Secuencia de Carbohidratos , Mananos/química , Mananos/inmunología , Ratones , Conejos , Albúmina Sérica/inmunología , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunologíaRESUMEN
A protective ß-mannan trisaccharide epitope from the Candida albicans cell wall phosphomannan has been synthesized and activated for copolymerization with acrylamide. The resulting glycopolymer displayed 33 trisaccharide haptens and was derivatized for conjugation to the immunogenic carrier protein, chicken serum albumin. The resulting conjugate achieves a high degree of oligosaccharide substitution while limiting the sites of substitution on the protein. The murine immune response against this conjugate was compared with the response to a trisaccharide-tetanus toxoid conjugate vaccine. The glycopolymer was shown to induce a more robust immune response with higher trisaccharide-specific antibody titers and with a significantly larger proportion of responding mice developing antibodies that bound the target, native cell wall antigen of C. albicans.
Asunto(s)
Candida albicans/química , Epítopos/química , Glicoconjugados/síntesis química , Mananos/síntesis química , Trisacáridos/síntesis química , Acrilamida/química , Animales , Candida albicans/citología , Pollos , Epítopos/inmunología , Femenino , Glicoconjugados/química , Glicoconjugados/inmunología , Mananos/química , Mananos/inmunología , Ratones , Conformación Molecular , Estructura Molecular , Albúmina Sérica/química , Toxoide Tetánico/síntesis química , Toxoide Tetánico/química , Toxoide Tetánico/inmunología , Trisacáridos/química , Trisacáridos/inmunología , Vacunación , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunologíaRESUMEN
The human ABO(H) blood group antigens are produced by specific glycosyltransferase enzymes. An N-acetylgalactosaminyltransferase (GTA) uses a UDP-GalNAc donor to convert the H-antigen acceptor to the A antigen, whereas a galactosyltransferase (GTB) uses a UDP-galactose donor to convert the H-antigen acceptor to the B antigen. GTA and GTB differ only in the identity of four critical amino acid residues. Crystal structures at 1.8-1.32 A resolution of the GTA and GTB enzymes both free and in complex with disaccharide H-antigen acceptor and UDP reveal the basis for donor and acceptor specificity and show that only two of the critical amino acid residues are positioned to contact donor or acceptor substrates. Given the need for stringent stereo- and regioselectivity in this biosynthesis, these structures further demonstrate that the ability of the two enzymes to distinguish between the A and B donors is largely determined by a single amino acid residue.
