RESUMEN
Synthetic vaccines that induce T cell responses to peptide epitopes are a promising immunotherapy for both communicable and noncommunicable diseases. Stimulating strong and sustained T cell responses requires antigen delivery to appropriately activated antigen presenting cells (APCs). One way this can be accomplished is by chemically conjugating immunogenic peptide epitopes with α-galactosylceramide (α-GalCer), a glycolipid that acts as an immune adjuvant by inducing stimulatory interactions between APCs and type I natural killer T (NKT) cells. Here we investigate whether increasing the ratio of antigen:adjuvant improves antigen-specific T cell responses. A series of conjugate vaccines was prepared in which one, two, four, or eight copies of an immunogenic peptide were covalently attached to a modified form of α-GalCer via the poly(ethoxyethylglycinamide) dendron scaffold. Initial attempts to synthesize these multivalent conjugate vaccines involved attaching the bicyclo[6.1.0]non-4-yne (BCN) group to the adjuvant-dendron structure followed by strain-promoted azide-alkyne cycloaddition of the peptide. Although this approach was successful for preparing vaccines with either one or two peptide copies, the synthesis of vaccines requiring attachment of four or eight BCN groups suffered from low yields due to cyclooctyne degradation. Instead, conjugate vaccines containing up to eight peptide copies were readily achieved through oxime ligation with adjuvant-dendron constructs decorated with the 8-oxo-nonanoyl group. When evaluating T cell responses to vaccination in mice, we confirmed a significant advantage to conjugation over admixes of peptide and α-GalCer, regardless of the peptide to adjuvant ratio, but there was no advantage to increasing the number of peptides attached. However, it was notable that the higher ratio conjugate vaccines required lower levels of NKT cell activation to be effective, which could be a safety advantage for future vaccine candidates.
RESUMEN
There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the ß-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods.
Asunto(s)
Proteínas Bacterianas/química , Carbohidrato Epimerasas/química , Hexosaminas/química , Staphylococcus aureus/enzimología , Fosfatos de Azúcar/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Carbohidrato Epimerasas/genética , Catálisis , Hexosaminas/genética , Hexosaminas/metabolismo , Mutación Missense , Conformación Proteica en Lámina beta , Dominios Proteicos , Staphylococcus aureus/genética , Fosfatos de Azúcar/genética , Fosfatos de Azúcar/metabolismoRESUMEN
Benchtop nuclear magnetic resonance (NMR) spectroscopy is a useful tool for the rapid determination of the self-diffusion coefficient and the hydrodynamic radius of dendrons. The self-diffusion coefficients of the first four generations of poly ethoxy ethyl glycinamide (PEE-G) dendrons are measured by diffusion-ordered spectroscopy (DOSY) on a benchtop NMR equipped with diffusion gradient coils. The hydrodynamic radii of the dendrons are calculated via the Stokes-Einstein equation. The effects of solvent and pH are determined with the hydrodynamic radius increasing with generation and decreasing upon neutralization of an acidic solution. These measurements provide valuable information for biological and pharmaceutical applications of dendrons.
RESUMEN
Morquio A syndrome is an autosomal mucopolysaccharide storage disorder that leads to accumulation of keratan sulfate. Diagnosis of this disease can be aided by measuring the levels of keratan sulfate in the urine. This requires the liquid chromatography tandem mass spectrometry (LCMS/MS) measurement of sulfated N-acetyl-d-lactosamines in the urine after cleavage of the keratan sulfate with keratanase II. Quantification requires isotopically-labelled internal standards. The synthesis of these 13 C6 -labelled standards from 13 C6 -galactose and N-acetylglucosamine is described. The required protected disaccharide is prepared utilising a regioselective, high yielding ß-galactosylation of a partially protected glucosamine acceptor and an inverse addition protocol. Subsequent synthesis of the 13 C6 -labelled mono and disulfated N-acetyllactosamines was achieved in five and eight steps, respectively, from this intermediate to provide internal standards for the LCMS/MS quantification of keratan sulfate in urine.
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Acetilgalactosamina/análogos & derivados , Espectrometría de Masas/métodos , Técnicas de Diagnóstico Molecular/métodos , Acetilgalactosamina/síntesis química , Isótopos de Carbono/química , Sulfato de Queratano/análisis , Sulfato de Queratano/orina , Mucopolisacaridosis IV/orinaRESUMEN
Heparan sulfate (HS) is a highly sulfated natural carbohydrate that plays crucial roles in cancer, inflammation, and angiogenesis. Heparanase (HPSE) is the sole HS degrading endoglycosidase that cleaves HS at structure-dependent sites along the polysaccharide chain. Overexpression of HPSE by cancer cells correlates with increased tumor size and enhanced metastasis. Previously we have shown that a tetramer HS mimetic is a potent HPSE inhibitor displaying remarkable anticancer activity inâ vivo. Building on that work, we report the synthesis and testing of a novel library of single entity trimer glycolipid mimetics that effectively inhibit HPSE at low nanomolar concentrations. A lipophilic arm was introduced to assess whether an improvement of pharmacokinetics and plasma residence time would offset the reduction in charge and multivalency. Preclinical tests in a mouse syngeneic model showed effective tumor growth inhibition by the tetramer but not the trimer glycomimetic.
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Neoplasias Colorrectales , Glucolípidos , Animales , Neoplasias Colorrectales/tratamiento farmacológico , Glucolípidos/farmacología , Heparitina Sulfato/farmacología , Ratones , Neovascularización PatológicaRESUMEN
The synthesis of analogues of natural enzyme substrates can be used to help deduce enzymatic mechanisms. N-Acetylmannosamine-6-phosphate 2-epimerase is an enzyme in the bacterial sialic acid catabolic pathway. To investigate whether the mechanism of this enzyme involves a re-protonation mechanism by the same neighbouring lysine that performed the deprotonation or a unique substrate-assisted proton displacement mechanism involving the substrate C5 hydroxyl, the syntheses of two analogues of the natural substrate, N-acetylmannosamine-6-phosphate, are described. In these novel analogues, the C5 hydroxyl has been replaced with a proton and a methyl ether respectively. As recently reported, Staphylococcus aureus N-acetylmannosamine-6-phosphate 2-epimerase was co-crystallized with these two compounds. The 5-deoxy variant bound to the enzyme active site in a different orientation to the natural substrate, while the 5-methoxy variant did not bind, adding to the evidence that this enzyme uses a substrate-assisted proton displacement mechanism. This mechanistic information may help in the design of potential antibacterial drug candidates.
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Proteínas Bacterianas/metabolismo , Carbohidrato Epimerasas/metabolismo , Hexosaminas/biosíntesis , Fosfatos de Azúcar/biosíntesis , Proteínas Bacterianas/química , Conformación de Carbohidratos , Carbohidrato Epimerasas/química , Hexosaminas/química , Staphylococcus aureus/enzimología , Fosfatos de Azúcar/químicaRESUMEN
A significant proportion of genetic disease cases arise from truncation of proteins caused by premature termination codons. In eukaryotic cells some aminoglycosides cause readthrough of premature termination codons during protein translation. Inducing readthrough of these codons can potentially be of therapeutic value in the treatment of numerous genetic diseases. A significant drawback to the repeated use of aminoglycosides as treatments is the lack of balance between their readthrough efficacy and toxicity. The synthesis and biological testing of designer aminoglycoside compounds is documented herein. We disclose the implementation of a strategy to reduce cellular toxicity and maintain readthrough activity of a library of compounds by modification of the overall cationic charge of the aminoglycoside scaffold through ring I modifications.
RESUMEN
The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.
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Prueba de Ácido Nucleico para COVID-19 , COVID-19 , Humanos , Indicadores y Reactivos/provisión & distribución , SARS-CoV-2RESUMEN
A range of poly ethoxy ethyl glycinamide (PEE-G) dendron scaffolds with gadolinium (III) complexes attached were synthesized with a focus on product purity and high Gd(III) loading. The nuclear magnetic resonance relaxivity of these products was measured and compared with commercially available low-molecular-weight magnetic resonance imaging contrast agents. Over twice the relaxivity based on Gd(III) concentration, and up to 20-fold increase in relaxivity were observed based on molecular concentration. Relaxivity properties were observed to increase with both increasing molecular weight and number of Gd(III) complexes attached, however a plateau was reached for molecular weight increase. T1 and T2 relaxivity properties were also investigated at two different magnetic fields. Transverse relaxivity is unaffected by magnetic field strength whereas increase in longitudinal relaxivity was not as pronounced at the higher field.
RESUMEN
Multivalent structures can provide multiple interactions at a target site and improve binding affinity. The multivalent presentation of the anti-tumour heptapeptide, SNTSESF, was investigated. This peptide's activity has been attributed to blockade of the PD-1 receptor-mediated signalling pathway. Two and four peptide units were conjugated to poly ethoxy ethyl glycinamide (PEE-G) scaffolds to prepare high-purity products. These conjugates and the peptide were examined in a mouse model implanted with GL261 tumours that indicated that presenting more than two copies of peptide SNTSESF on the dendritic scaffold does not increase anti-tumour activity per peptide. The fluorescent labelled peptide and most active multivalent peptide conjugate were therefore screened for their interaction with the human PD-L1 protein in a fluorescence polarisation assay. No indication of a specific SNTSESF peptide/PD-L1 interaction was observed. This finding was further supported by a molecular modelling binding study.
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Glicina/análogos & derivados , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Glicina/síntesis química , Glicina/química , Glicina/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Receptor de Muerte Celular Programada 1/metabolismo , Relación Estructura-ActividadRESUMEN
Immunostimulatory saponin based colloidal antigen delivery systems show promise as adjuvants for subunit vaccines. For this reason, allyl oleanolate was glycosylated at the 3-position using trichloroacetimidate donors to give monodesmodic saponins following deprotection. Bisdesmodic saponins were synthesized by double glycosylation at the 3- and 28-positions of oleanolic acid. When formulated together with cholesterol and phospholipids, ring-like, helical and rod-like nanostructures were formed depending on the saponin concentrations used. As an indication of adjuvant activity, the ability of these formulations, and the saponins by themselves, to induce dendritic cell maturation was measured, but no significant activity was observed.
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Ácido Glicirrínico/química , ISCOMs/química , ISCOMs/farmacología , Ácido Oleanólico/química , Saponinas/química , Saponinas/farmacología , Animales , Colesterol/química , Células Dendríticas/citología , Glicosilación , Hematopoyesis/efectos de los fármacos , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Nanoestructuras/química , Fosfolípidos/químicaRESUMEN
The synthesis of Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-acetyl)-alpha-d-mannopyranosyl]-l-proline allyl ester and Nalpha-fluorenylmethoxycarbonyl-trans-4-hydroxy-4-O-[(2,3,4,6-tetra-O-benzoyl)-alpha-d-mannopyranosyl]-l-proline allyl ester is described. Glycosylation using Königs-Knorr conditions with a benzoyl protected glycosyl donor provided the optimum method. Removal of the allyl ester gave two mannosylated building blocks suitable for solid phase glycopeptide synthesis.
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Química/métodos , Fluorenos/síntesis química , Prolina/análogos & derivados , Prolina/química , Piranos/química , Aminoácidos/química , Conformación de Carbohidratos , Carbohidratos/química , Ésteres/química , Glicosilación , Lectinas/química , Espectroscopía de Resonancia Magnética , Manosa/química , Modelos Químicos , Prolina/síntesis química , Espectrofotometría/métodosRESUMEN
Poly ethoxy ethyl glycinamide (PEE-G) dendrimers have been specifically designed and synthesized with the aim of providing a readily available dendrimer scaffold that can be used to make products that can meet the stringent requirements of pharmaceutical applications. The synthesis has been refined to produce dendrimers that are of high HPLC purity. The suitability of PEE-G dendrimers for their designed use has been verified by subsequent measurements to demonstrate that they are of high stability, high aqueous solubility, low cytotoxicity, low immunogenicity and with low inâ vivo toxicity in an escalating-dose rat study. PEE-G dendrimers therefore provide a useful scaffold for researchers wanting to develop dendrimer-based drug candidates.
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Dendrímeros/síntesis química , Animales , Supervivencia Celular/efectos de los fármacos , Dendrímeros/toxicidad , Descubrimiento de Drogas , Femenino , Masculino , Ratones Endogámicos C57BL , Ratas Sprague-Dawley , Ovinos , Bazo/citología , Bazo/efectos de los fármacosRESUMEN
OBJECTIVE: Morquio A syndrome (mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by deficient N-acetylgalactosamine-6-sulphatase (GALNS) activity. Early and accurate diagnosis of this condition is critical for improved patient outcomes, particularly as enzyme replacement therapy has recently become available. An LC-MS/MS assay utilising keratan sulphate (KS) disaccharides derived from keratanase-II digestion provides a sensitive and specific means for quantitation of urinary KS, a screening biomarker for Morquio A (Oguma et al., 2007; Martell et al., 2011). To ensure a reliable supply of keratanase-II, we sought to produce a Bacillus circulans-derived enzyme via a recombinant approach in Escherichia coli. DESIGN AND METHODS: Bioinformatics analysis of the B. circulans keratanase-II enzyme identified likely dispensable C-terminal domains amenable to enhancement via protein engineering. A truncated form of the enzyme was designed to remove the domains predicted to be unnecessary for catalytic activity and detrimental to recombinant expression in E. coli. RESULTS: C-terminally truncated, recombinant B. circulans keratanase-II was purified to >98% homogeneity and extensively characterised, demonstrating desired activity, specificity and utility in LC-MS-based quantitation of urinary KS from Morquio A and control samples, and is functionally indistinguishable from full-length, native B. circulans-derived keratanase-II. CONCLUSIONS: This novel, recombinant keratanase-II meets all performance requirements and can be produced in a rapid and reproducible manner. We speculate that other related bacterial enzymes of biomedical or industrial interest may be amenable to similar engineered enhancements.
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Acetilglucosaminidasa/química , Sulfato de Queratano/orina , Mucopolisacaridosis IV/orina , Acetilglucosaminidasa/biosíntesis , Acetilglucosaminidasa/genética , Acetilglucosaminidasa/metabolismo , Adolescente , Adulto , Animales , Bacillus/enzimología , Bacillus/genética , Bioingeniería/métodos , Biomarcadores/orina , Estudios de Casos y Controles , Catálisis , Bovinos , Niño , Preescolar , Cromatografía Liquida/métodos , Clonación Molecular , Escherichia coli/enzimología , Escherichia coli/genética , Humanos , Estructura Terciaria de Proteína , Espectrometría de Masas en Tándem/métodos , Adulto JovenRESUMEN
In an attempt to discover a new synthetic vaccine adjuvant, the glycosylation of hederagenin, gypsogenin, and oleanolic acid acceptors with di- and trisaccharide donors to generate a range of mimics of natural product QS-21 was carried out. The saponins were formulated with phosphatidylcholine and cholesterol, and the structures analyzed by transmission electron microscopy. 3-O-(Manp(1â3)Glcp)hederagenin was found to produce numerous ring-like micelles when formulated, while C-28 choline ester derivatives preferred self-assembly and did not interact with the liposomes. When alone and in the presence of cholesterol and phospholipid, the choline ester derivatives produced nanocrystalline rods or helical micelles. The effects of modifying sugar stereochemistry and the aglycone on the immunostimulatory effects of the saponins was then evaluated using the activation markers MHC classâ II and CD86 in murine bone marrow dendritic cells. The most active saponin, 3-O-(Manp(1â3)Glcp)hederagenin, was stimulatory at high concentrations in cell culture, but this did not translate to strong responses in vivo.
RESUMEN
Readily available, crystalline penta-O-propanoyl-beta-D-glucofuranose is shown to be a suitable glycosylating agent for the acid-catalysed, direct synthesis of O-, S- and N-glucofuranosyl compounds. Beta-linked products are formed with good selectivity. Reaction with cyanotrimethylsilane gave the 1,2-O-(1-cyanopropylidene)acetal rather than the C-glycosyl cyanide. By selective acid-catalysed hydrolysis, the title compound was converted to the 1-hydroxy analogue from which the trichloroacetimidates were made as further potential glycosylating agents.
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Cloroacetatos , Glucósidos/química , Acetamidas , Furanos , Glicosilación , Hidrólisis , Espectroscopía de Resonancia MagnéticaRESUMEN
A chromogenic substrate, 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate was synthesized and used in combination with beta-N-acetylhexosaminidase for detection of the sulfatase, MdsA, by release of 4-nitrophenol. MdsA was originally isolated from the bacterium Prevotella strain RS2 and is believed to be involved in desulfation of sulfomucins, major components of the mucus barrier protecting the human colon surface. The exo nature of the MdsA sulfatase was indicated by its inability to de-esterify the disaccharide 4-nitrophenyl beta-D-galactopyranosyl-(1-->4)-2-acetamido-2-deoxy-beta-D-glucopyranoside 6-sodium sulfate. This latter compound was prepared from monosaccharide precursors by two different methods, the shorter requiring just six steps from 4-nitrophenyl 2-acetamido-2-deoxy-beta-D-glucopyranoside and giving an overall yield of 26.4%. The syntheses of 4-nitrophenyl beta-D-galactopyranoside 3-triethylammonium sulfate and 6-triethylammonium sulfate and their use in combination with beta-galactosidase as chromogenic substrates for detecting Bacteroides fragilis sulfatases with different specificities was also demonstrated.
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Compuestos Cromogénicos/síntesis química , Mucinas/metabolismo , Oligosacáridos/síntesis química , Sulfatasas/metabolismo , Sulfatos/síntesis química , Aspergillus oryzae/enzimología , Bacteroides fragilis/enzimología , Compuestos Cromogénicos/química , Modelos Químicos , Estructura Molecular , Mucinas/química , Oligosacáridos/química , Sulfatos/químicaRESUMEN
Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in ß-defensin, IL-10, interferon-ß, transforming growth factor-ß, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases.
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Dendrímeros/administración & dosificación , Disentería Bacilar/tratamiento farmacológico , Fármacos Gastrointestinales/administración & dosificación , Tracto Gastrointestinal/efectos de los fármacos , Glucosamina/análogos & derivados , Interleucina-6/antagonistas & inhibidores , Interleucina-8/antagonistas & inhibidores , Administración Oral , Animales , Traslocación Bacteriana/efectos de los fármacos , Diarrea/tratamiento farmacológico , Diarrea/patología , Modelos Animales de Enfermedad , Disentería Bacilar/patología , Tracto Gastrointestinal/patología , Glucosamina/administración & dosificación , Factores Inmunológicos/administración & dosificación , Conejos , Shigella/patogenicidadRESUMEN
The structure of the ternary complex of mycothiol synthase from Mycobacterium tuberculosis with bound desacetylmycothiol and CoA was determined to 1.8 A resolution. The structure of the acetyl-CoA-binary complex had shown an active site groove that was several times larger than its substrate. The structure of the ternary complex reveals that mycothiol synthase undergoes a large conformational change in which the two acetyltransferase domains are brought together through shared interactions with the functional groups of desacetylmycothiol, thereby decreasing the size of this large central groove. A comparison of the binary and ternary structures illustrates many of the features that promote catalysis. Desacetylmycothiol is positioned with its primary amine in close proximity and in the proper orientation for direct nucleophilic attack on the si-face of the acetyl group of acetyl-CoA. Glu-234 and Tyr-294 are positioned to act as a general base and general acid to promote acetyl transfer. In addition, this structure provides further evidence that the N-terminal acetyltransferase domain no longer has enzymatic activity and is vestigial in nature.