RESUMEN
UDP-galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non-substrate-like inhibitor, MS-208, with M.â tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS-208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS-208 binds at an allosteric binding site (A-site) instead of the enzyme active site (S-site). A candidate A-site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A-site. Further molecular dynamics studies led us to propose that MS-208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A-site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Transferasas Intramoleculares/antagonistas & inhibidores , Mycobacterium tuberculosis/enzimología , Pirazoles/farmacología , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Transferasas Intramoleculares/metabolismo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Molecular , Pirazoles/química , Relación Estructura-ActividadRESUMEN
Influenza virus neuraminidase (NA) drug resistance is one of the challenges to preparedness against epidemic and pandemic influenza virus infections. NA N1- and N2-containing influenza viruses are the primary cause of seasonal epidemics and past pandemics. The structural and functional basis underlying drug resistance of the influenza virus N1 NA is well characterized. Yet drug resistance of the N2 strain is not well understood. Here, we confirm that replacement of N2 E119 or I222 results in multidrug resistance, and when the replacements occur together, the sensitivity to NA inhibitors (NAI) is reduced severely. Using crystallographic studies, we showed that E119 replacement results in a loss of hydrogen bonding to oseltamivir and zanamivir, whereas I222 replacement results in a change in the hydrophobic environment that is critical for oseltamivir binding. Moreover, we found that MS-257, a zanamivir-oseltamivir hybrid inhibitor, is less susceptible to drug resistance. The binding mode of MS-257 shows that increased hydrogen bonding interactions between the inhibitor and NA active site anchor the inhibitor within the active site and allow adjustments in response to active-site modifications. Such stability is likely responsible for the observed reduced susceptibility to drug resistance. MS-257 serves as a next-generation anti-influenza virus drug candidate and serves also as a scaffold for further design of NAIs. IMPORTANCE: Oseltamivir and zanamivir are the two major antiviral drugs available for the treatment of influenza virus infections. However, multidrug-resistant viruses have emerged in clinical cases, which pose a challenge for the development of new drugs. N1 and N2 subtypes exist in the viruses which cause seasonal epidemics and past pandemics. Although N1 drug resistance is well characterized, the molecular mechanisms underlying N2 drug resistance are unknown. A previous report showed that an N2 E119V/I222L dual mutant conferred drug resistance to seasonal influenza virus. Here, we confirm that these substitutions result in multidrug resistance and dramatically reduced sensitivity to NAI. We further elucidate the molecular mechanism underlying N2 drug resistance by solving crystal structures of the N2 E119V and I222L mutants and the dual mutant. Most importantly, we found that a novel oseltamivir-zanamivir hybrid inhibitor, MS-257, remains more effective against drug-resistant N2 and is a promising candidate as a next-generation anti-influenza virus drug.
Asunto(s)
Antivirales/farmacología , Virus de la Influenza A/efectos de los fármacos , Virus de la Influenza A/enzimología , Mutación , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Oseltamivir/farmacología , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética , Zanamivir/farmacología , Sustitución de Aminoácidos , Farmacorresistencia Viral Múltiple/genética , Inhibidores Enzimáticos/farmacología , Humanos , Virus de la Influenza A/genética , Modelos Moleculares , Neuraminidasa/química , Proteínas Virales/químicaRESUMEN
This manuscript describes a novel class of derivatives based on a bicyclo[3.1.0]hexane scaffold, proposed as mimics of sialic acid in a distorted boat conformation that is on the catalytic pathway of neuraminidases (sialidases). A general synthetic route for these constrained-ring molecules was developed using a photochemical reaction followed by a Johnson-Corey-Chaykovsky cyclopropanation. Functionalization with the goal of occupying the 150-cavity was also exploited. Inhibition assays demonstrated low micromolar inhibition against both group-1 (H5N1) and group-2 (H9N2) influenza neuraminidase subtypes, indicating good affinity for the alpha and beta sialic acid mimics and 150-cavity-targeted derivatives. These results provide a validation of a bicyclo[3.1.0]hexane scaffold as a mimic of a distorted sialic acid bound in the neuraminidase active site during catalysis.
Asunto(s)
Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Subtipo H5N1 del Virus de la Influenza A/enzimología , Subtipo H9N2 del Virus de la Influenza A/enzimología , Neuraminidasa/antagonistas & inhibidores , Compuestos Bicíclicos con Puentes/química , Técnicas de Química Sintética , Diseño de Fármacos , Inhibidores Enzimáticos/química , Modelos Moleculares , Neuraminidasa/química , Conformación ProteicaRESUMEN
The primary sweet sensor in mammalian taste cells for sugars and noncaloric sweeteners is the heteromeric combination of type 1 taste receptors 2 and 3 (T1R2+T1R3, encoded by Tas1r2 and Tas1r3 genes). However, in the absence of T1R2+T1R3 (e.g., in Tas1r3 KO mice), animals still respond to sugars, arguing for the presence of T1R-independent detection mechanism(s). Our previous findings that several glucose transporters (GLUTs), sodium glucose cotransporter 1 (SGLT1), and the ATP-gated K(+) (KATP) metabolic sensor are preferentially expressed in the same taste cells with T1R3 provides a potential explanation for the T1R-independent detection of sugars: sweet-responsive taste cells that respond to sugars and sweeteners may contain a T1R-dependent (T1R2+T1R3) sweet-sensing pathway for detecting sugars and noncaloric sweeteners, as well as a T1R-independent (GLUTs, SGLT1, KATP) pathway for detecting monosaccharides. However, the T1R-independent pathway would not explain responses to disaccharide and oligomeric sugars, such as sucrose, maltose, and maltotriose, which are not substrates for GLUTs or SGLT1. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we found that taste cells express multiple α-glycosidases (e.g., amylase and neutral α glucosidase C) and so-called intestinal "brush border" disaccharide-hydrolyzing enzymes (e.g., maltase-glucoamylase and sucrase-isomaltase). Treating the tongue with inhibitors of disaccharidases specifically decreased gustatory nerve responses to disaccharides, but not to monosaccharides or noncaloric sweeteners, indicating that lingual disaccharidases are functional. These taste cell-expressed enzymes may locally break down dietary disaccharides and starch hydrolysis products into monosaccharides that could serve as substrates for the T1R-independent sugar sensing pathways.
Asunto(s)
Disacáridos/farmacología , Regulación Enzimológica de la Expresión Génica/fisiología , Papilas Gustativas/enzimología , Gusto/fisiología , alfa-Glucosidasas/biosíntesis , Animales , Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Ratones , Ratones Transgénicos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transportador 1 de Sodio-Glucosa/genética , Transportador 1 de Sodio-Glucosa/metabolismo , alfa-Glucosidasas/genéticaRESUMEN
Uridine diphosphate-galactopyranose mutase (UGM), an enzyme found in many eukaryotic and prokaryotic human pathogens, catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf), the latter being used as the biosynthetic precursor of the galactofuranose polymer portion of the mycobacterium cell wall. We report here the synthesis of a sulfonium and selenonium ion with an appended polyhydroxylated side chain. These compounds were designed as transition state mimics of the UGM-catalyzed reaction, where the head groups carrying a permanent positive charge were designed to mimic both the shape and positive charge of the proposed galactopyranosyl cation-like transition state. An HPLC-based UGM inhibition assay indicated that the compounds inhibited about 25% of UGM activity at 500 µM concentration.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/farmacología , Galactosa/análogos & derivados , Isomerasas/antagonistas & inhibidores , Uridina Difosfato/análogos & derivados , Biocatálisis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Galactosa/metabolismo , Hidroxilación , Isomerasas/metabolismo , Mycobacterium tuberculosis/enzimología , Compuestos de Selenio/síntesis química , Compuestos de Selenio/química , Compuestos de Selenio/farmacología , Compuestos de Sulfonio/síntesis química , Compuestos de Sulfonio/química , Compuestos de Sulfonio/farmacología , Uridina Difosfato/metabolismoRESUMEN
A practical method for simultaneously quantifying fructose and ethanol contents in wines using (13)C quantitative nuclear magnetic resonance (qNMR) spectroscopy is reported. Less than 0.6 mL of wine is needed, and the method leaves an unmodified sample available for subsequent testing or additional analyses. The relative ratios of the five known fructose isomers in ethanolic solutions at different pH and their variations with the temperature are also reported. The data are correlated with the sweetness of wines. The technique was applied to commercially available wines, and the results are compared to other methods. Sugar levels above 0.6 g/L can also be measured. A simple adaptation of the method permits measurement of different carbohydrates using integration of single peaks for each compound, in combination with an external reference (13)C qNMR spectrum of a sample with a known concentration. The method can be applied at all stages of wine production, including grape must, during fermentation, and before and after bottling.
Asunto(s)
Fructosa/química , Espectroscopía de Resonancia Magnética/métodos , Vino/análisis , Isomerismo , Estructura MolecularRESUMEN
The synthesis of 1-[5-O-(α-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione and 1-[(5-O-(ß-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione as non-ionic substrate mimics of UDP-Galp are described. UDP-Galp is a precursor of Galf, which is a primary component of the cell-wall glycans of several microorganisms. The interconversion of UDP-Galp and UDP-Galf is catalyzed by UDP galactopyranose mutase (UGM); its inhibition comprises a mode of compromising the microorganisms. The nonionic polyhydroxylated chain was intended to mimic the ionic pyrophosphate group and the ribose moiety in UDP-Galp and increase the bioavailabilities of the candidate inhibitors. Inhibition assays with UGM of Mycobacterium tuberculosis showed only weak inhibition of the enzyme by these compounds.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Galactosa/metabolismo , Transferasas Intramoleculares/antagonistas & inhibidores , Monosacáridos/farmacología , Uridina Difosfato/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Transferasas Intramoleculares/metabolismo , Conformación Molecular , Monosacáridos/síntesis química , Monosacáridos/química , Mycobacterium tuberculosis/enzimología , Relación Estructura-ActividadRESUMEN
UDP-galactopyranose mutase (UGM) is an enzyme involved in the biosynthesis of the Mycobacterium tuberculosis cell wall, and is essential for the growth and survival of the organism. A micromolar inhibitor developed by tetrafluorination of the UGM substrate has been previously studied by saturation transfer difference (STD) NMR spectroscopy. To elucidate the bioactive conformation of the inhibitor bound to UGM, we employ molecular dynamics (MD) simulations to construct a structural model. The MD model is subsequently validated by a good fit between experimental and theoretical STD effects, the latter calculated by a complete relaxation and conformational exchange matrix (CORCEMA) analysis. This structural model is used to explain the relative binding affinities of the inhibitor and the parent substrate.
Asunto(s)
Inhibidores Enzimáticos/química , Transferasas Intramoleculares/antagonistas & inhibidores , Simulación de Dinámica Molecular , Sitios de Unión , Inhibidores Enzimáticos/metabolismo , Transferasas Intramoleculares/metabolismo , Klebsiella pneumoniae/enzimología , Espectroscopía de Resonancia Magnética , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.
Asunto(s)
N-Acetilgalactosaminiltransferasas/química , Conformación Proteica , Especificidad por Sustrato , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Lactate dehydrogenase A (LDHA) is an important enzyme in fermentative glycolysis, generating most energy for cancer cells that rely on anaerobic respiration even under normal oxygen concentrations. This renders LDHA a promising molecular target for the treatment of various cancers. Several efforts have been made recently to develop LDHA inhibitors with nanomolar inhibition and cellular activity, some of which have been studied in complex with the enzyme by X-ray crystallography. In this work, we present a molecular dynamics (MD) study of the binding interactions of selected ligands with human LDHA. Conventional MD simulations demonstrate different binding dynamics of inhibitors with similar binding affinities, whereas steered MD simulations yield discrimination of selected LDHA inhibitors with qualitative correlation between the in silico unbinding difficulty and the experimental binding strength. Further, our results have been used to clarify ambiguities in the binding modes of two well-known LDHA inhibitors.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , L-Lactato Deshidrogenasa/antagonistas & inhibidores , Simulación de Dinámica Molecular , Sitios de Unión , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , L-Lactato Deshidrogenasa/química , L-Lactato Deshidrogenasa/metabolismo , Lactato Deshidrogenasa 5 , Ligandos , Estructura Secundaria de Proteína , Especificidad por Sustrato , TermodinámicaRESUMEN
We have previously reported a potent neuraminidase inhibitor that comprises a carbocyclic analogue of zanamivir in which the hydrophilic glycerol side chain is replaced by the hydrophobic 3-pentyloxy group of oseltamivir. This hybrid inhibitor showed excellent inhibitory properties in the neuraminidase inhibition assay (Ki =0.46â nM; Ki (zanamivir) =0.16â nM) and in the viral replication inhibition assay in cell culture at 10(-8) M. As part of this lead optimization, we now report a novel spirolactam that shows comparable inhibitory activity in the cell culture assay to that of our lead compound at 10(-7) M. The compound was discovered serendipitously during the attempted synthesis of the isothiourea derivative of the original candidate. The X-ray crystal structure of the spirolactam in complex with the N8 subtype neuraminidase offers insight into the mode of inhibition.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Lactamas/farmacología , Neuraminidasa/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Proteínas Virales/antagonistas & inhibidores , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Lactamas/síntesis química , Lactamas/química , Modelos Moleculares , Conformación Molecular , Neuraminidasa/metabolismo , Compuestos de Espiro/síntesis química , Compuestos de Espiro/química , Relación Estructura-Actividad , Proteínas Virales/metabolismoRESUMEN
Pyranose-furanose mutases are essential enzymes in the life cycle of a number of microorganisms, but are absent in mammalian systems, and hence represent novel targets for drug development. To date, all such mutases show preferential recognition of a single substrate (e.g., UDP-Gal). We report here the detailed structural characterization of the first bifunctional pyranose-furanose mutase, which recognizes both UDP-Gal and UDP-GalNAc. The enzyme under investigation (cjUNGM) is involved in the biosynthesis of capsular polysaccharides (CPSs) in Campylobacter jejuni 11168. These CPSs are known virulence factors that are required for adhesion and invasion of human epithelial cells. Using a combination of UV/visible spectroscopy, X-ray crystallography, saturation transfer difference NMR spectroscopy, molecular dynamics and CORCEMA-ST calculations, we have characterized the binding of the enzyme to both UDP-Galp and UDP-GalpNAc, and compared these interactions with those of a homologous monofunctional mutase enzyme from E. coli (ecUGM). These studies reveal that two arginines in cjUNGM, Arg59 and Arg168, play critical roles in the catalytic mechanism of the enzyme and in controlling its specificity to ultimately lead to a GalfNAc-containing CPS. In ecUGM, these arginines are replaced with histidine and lysine, respectively, and this results in an enzyme that is selective for UDP-Gal. We propose that these changes in amino acids allow C. jejuni 11168 to produce suitable quantities of the sugar nucleotide substrate required for the assembly of a CPS containing GalfNAc, which is essential for viability.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Campylobacter/terapia , Campylobacter jejuni/enzimología , Transferasas Intramoleculares/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , Biocatálisis , Infecciones por Campylobacter/metabolismo , Infecciones por Campylobacter/patología , Cristalografía por Rayos X , Escherichia coli/enzimología , Humanos , Transferasas Intramoleculares/química , Simulación de Dinámica Molecular , Unión Proteica , Estructura Terciaria de Proteína , Especificidad por Sustrato , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato N-Acetilgalactosamina/química , Uridina Difosfato N-Acetilgalactosamina/metabolismoRESUMEN
In humans, four different enzymes mediate the digestion of ingested carbohydrates. First salivary and pancreatic α-amylases, the two endoacting retaining glucosidases, break down the complex starch molecules into smaller linear maltose-oligomers (LM) and branched α-limit dextrins (αLDx). Then two retaining exoglucosidases, maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI), convert those molecules into glucose in the small intestine. The small intestinal brush-border epithelial cells anchor MGAM and SI, and each contains a catalytic N- and C-terminal subunit, ntMGAM, ctMGAM, ntSI, and ctSI, respectively. All four catalytic domains have, to varying extents, α-1,4-exohydrolytic glucosidase activity and belong to the glycoside hydrolase family 31 (GH31). ntSI and ctSI show additional activity toward α-1,6 (isomaltose substrates) and α-1,2 (sucrose) glycosidic linkages, respectively. Because they mediate the final steps of starch digestion, both MGAM and SI are important target enzymes for the treatment of type-2 diabetes. Because of their potent inhibitory activities against the mammalian intestinal α-glucosidases, sulfonium-ion glucosidase inhibitors isolated from the antidiabetic herbal extracts of various Salacia species have received considerable attention recently. Thus far, researchers have isolated eight sulfonium-ion glucosidase inhibitors from Salacia species: salaprinol, salacinol, ponkoranol, kotalanol, and four of their corresponding de-O-sulfonated compounds, the structures of which comprise a 1,4-anhydro-4-thio-d-arabinitol and a polyhydroxylated acyclic side chain. Some of these compounds more strongly inhibit human intestinal α-glucosidases than the currently available antidiabetic drugs, acarbose and miglitol, and could serve as lead candidates in the treatment of type-2 diabetes. In this Account, we summarize progress in the field since 2010 with this class of inhibitors, with particular focus on their selective inhibitory activities against the intestinal glucosidases. Through structure-activity relationship (SAR) studies, we have modified the natural compounds to derive more potent, nanomolar inhibitors of human MGAM and SI. This structural optimization also yielded the most potent inhibitors known to date for each subunit. Furthermore, we observed that some of our synthetic inhibitors selectively blocked the activity of some mucosal α-glucosidases. Those results led to our current working hypothesis that selective inhibitors can dampen the action of a fast digesting subunit or subunits which places the burden of digestion on slower digesting subunits. That strategy can control the rate of starch digestion and glucose release to the body. Decreasing the initial glucose spike after a carbohydrate-rich meal and extending postprandial blood glucose delivery to the body can be desirable for diabetics and patients with other metabolic syndrome-associated diseases.
Asunto(s)
Productos Biológicos/farmacología , Inhibidores Enzimáticos/farmacología , Glucosidasas/antagonistas & inhibidores , Hipoglucemiantes/farmacología , Compuestos de Azufre/farmacología , Animales , Productos Biológicos/química , Inhibidores Enzimáticos/química , Humanos , Hipoglucemiantes/química , Relación Estructura-Actividad , Compuestos de Azufre/químicaRESUMEN
The synthesis and immunogenicity of a tetanus toxoid (TT)-conjugate of the hexasaccharide portion of the cell-wall polysaccharide (CWPS) of the Group A Streptococcus (GAS) is described. The synthesis relies on the reaction of an allyl glycoside of the hexasaccharide with cysteamine, followed by the reaction of the resultant amine with diethyl squarate to give the monoethyl squarate adduct. Subsequent reaction with the lysine ε-amino groups on TT gives the glycoconjugate containing 30 hexasaccharide haptens per TT molecule. The immunogenicity in mice is similar to that obtained with a native CWPS-TT conjugate, validating the glycoconjugate as a vaccine candidate against GAS infections.
Asunto(s)
Inmunoconjugados/química , Inmunoconjugados/inmunología , Polisacáridos Bacterianos/síntesis química , Polisacáridos Bacterianos/inmunología , Vacunas Estreptocócicas/síntesis química , Vacunas Estreptocócicas/inmunología , Animales , Formación de Anticuerpos , Secuencia de Carbohidratos , Femenino , Inmunoconjugados/farmacología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Polisacáridos Bacterianos/farmacología , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/farmacología , Toxoide Tetánico/síntesis química , Toxoide Tetánico/inmunologíaRESUMEN
We report here the exploitation of the 150 cavity in the active site of influenza A viral neuraminidases for the design of novel C-6 triazole-containing Tamiflu derivatives. A general and convenient synthetic route was developed by utilizing a highly substituted cyclic Baylis-Hillman acetate as an active precursor for azide substitution via suprafacial allylic azide [3,3]-sigmatropic rearrangement. Virus replication inhibitory assays in vitro of these triazole derivatives containing either an amino or guanidino function indicated that the guanidinium compound showed the higher efficacy against a strain with N2 subtype at a concentration of 2 × 10(-5) M but did not inhibit replication of a strain with N1 subtype even at a concentration of 10(-4) M. In order to probe the nature of the enzyme-inhibitor interactions, molecular dynamics simulations were performed on complexes of these compounds with different neuraminidase enzymes. The results indicated that the candidate inhibitors occupy both the 150 cavity and catalytic site but with alternating occupancy.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Virus de la Influenza A/química , Virus de la Influenza A/enzimología , Neuraminidasa/química , Oseltamivir/química , Oseltamivir/síntesis química , Dominio Catalítico , Diseño de Fármacos , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/metabolismo , Oseltamivir/análogos & derivados , Triazoles/químicaRESUMEN
The influenza virus neuraminidase (NA) is essential for the virus life cycle. The rise of resistance mutations against current antiviral therapies has increased the need for the development of novel inhibitors. Recent efforts have targeted a cavity adjacent to the catalytic site (the 150-cavity) in addition to the primary catalytic subsite in order to increase specificity and reduce the likelihood of resistance. This study details structural and in vitro analyses of a class of inhibitors that bind uniquely in both subsites. Crystal structures of three inhibitors show occupation of the 150-cavity in two distinct and novel binding modes. We believe these are the first nanomolar inhibitors of NA to be characterized in this way. Furthermore, we show that one inhibitor, binding within the catalytic site, offers reduced susceptibility to known resistance mutations via increased flexibility of a pendant pentyloxy group and the ability to pivot about a strong hydrogen-bonding network.
Asunto(s)
Antivirales/química , Inhibidores Enzimáticos/química , Virus de la Influenza A/enzimología , Neuraminidasa/química , Proteínas Virales/química , Animales , Antivirales/metabolismo , Antivirales/farmacología , Dominio Catalítico , Línea Celular , Farmacorresistencia Viral , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Virus de la Influenza A/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/metabolismo , Oseltamivir/química , Oseltamivir/farmacología , Unión Proteica , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismoRESUMEN
The synthesis of a tetanus toxoid (TT)-conjugate of a hyaluronic acid (HA) hexasaccharide is described. The compound was intended for use in monitoring HA levels as a disease marker and as a potential vaccine against Group A Streptococcus (GAS) infections. We also report the synthesis of a chemically modified HA-hexasaccharide-TT conjugate in which the N-acetyl moiety of the N-acetyl-D-glucosamine residue is replaced with an N-propionyl unit in order to enhance immunogenicity. The oligosaccharides are synthesized in a convergent manner. The TT-conjugate syntheses rely on the reaction of the amines on the 6-aminohexyl aglycon of the hexasaccharides with diethyl squarate to give the monoethyl squarate adducts. Subsequent reactions with lysine ε-amino groups on TT then give the glycoconjugates containing an average of 8 hexasaccharide haptens per TT molecule. Immunological studies in mice show very similar antibody responses with both conjugates, suggesting that the N-acetyl groups of the glucosaminyl residues of the HA-hexasaccharide are not a critical part of the epitope recognized by the anti-HA polyclonal immune response. Furthermore, it would appear that the N-acyl moieties are not in close contact with the amino acid residues of the antibody combining sites.
Asunto(s)
Ácido Hialurónico/inmunología , Oligosacáridos/inmunología , Infecciones Estreptocócicas/inmunología , Vacunas Estreptocócicas/inmunología , Streptococcus/inmunología , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/farmacología , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Oligosacáridos/química , Oligosacáridos/farmacología , Albúmina Sérica/química , Albúmina Sérica/inmunología , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/química , Vacunas Estreptocócicas/farmacología , Streptococcus/efectos de los fármacos , Toxoide Tetánico/química , Toxoide Tetánico/inmunologíaRESUMEN
Neuraminidase inhibitors are the main pharmaceutical agents employed for treatments of influenza infections. The neuraminidase structures typically exhibit a 150-cavity, an exposed pocket that is adjacent to the catalytic site. This site offers promising additional contact points for improving potency of existing pharmaceuticals, as well as generating entirely new candidate inhibitors. Several inhibitors based on known compounds and designed to interact with 150-cavity residues have been reported. However, the dynamics of any of these inhibitors remains unstudied and their viability remains unknown. This work reports the outcome of long-term, all-atom molecular dynamics simulations of four such inhibitors, along with three standard inhibitors for comparison. Each is studied in complex with four representative neuraminidase structures, which are also simulated in the absence of ligands for comparison, resulting in a total simulation time of 9.6 µs. Our results demonstrate that standard inhibitors characteristically reduce the mobility of these dynamic proteins, while the 150-binders do not, instead giving rise to many unique conformations. We further describe an improved RMSD-based clustering technique that isolates these conformations--the structures of which are provided to facilitate future molecular docking studies--and reveals their interdependence. We find that this approach confers many advantages over previously described techniques, and the implications for rational drug design are discussed.
Asunto(s)
Virus de la Influenza A/enzimología , Simulación de Dinámica Molecular , Neuraminidasa/metabolismo , Apoproteínas/química , Apoproteínas/metabolismo , Sitios de Unión , Dominio Catalítico , Análisis por Conglomerados , Estabilidad de Enzimas , Ligandos , Neuraminidasa/química , Estructura Secundaria de ProteínaRESUMEN
Humans have the highest level of adenosine-to-inosine (A-to-I) editing amongst primates, yet the reasons for this difference remain unclear. Sequence analysis of the Alu Sg elements (A-to-I RNA substrates) corresponding to the Nup50 gene in human, chimp, and rhesus reveals subtle sequence variations surrounding the edit sites. We have developed three constructs that represent human (HuAp5), chimp (ChAp5), and rhesus (RhAp5) Nup50 Alu Sg A-to-I editing substrates. Here, 2-aminopurine (2-Ap) was substituted for edited adenosine (A5) so as to monitor the fluorescence intensity with respect to temperature. UV and steady-state fluorescence (SSF) T(M) plots indicate that local and global unfolding are coincident, with the human construct displaying a T(M) of approximately 70°C, compared to 60°C for chimp and 54°C for rhesus. However, time-resolved fluorescence (TRF) resolves three different fluorescence lifetimes that we assign to folded, intermediate(s), and unfolded states. The TRF data fit well to a two-intermediate model, whereby both intermediates (M, J) are in equilibrium with each other, and the folded/unfolded states. Our model suggests that, at 37°C, human state J and the folded state will be the most heavily populated in comparison to the other primate constructs. In order for adenosine deaminase acting on RNA (ADAR) to efficiently dock, a stable duplex must be present that corresponds to the human construct, globally. Next, the enzyme must "flip out" the base of interest to facilitate the A-to-I conversion; a nucleotide in an intermediate-like position would enhance this conformational change. Our experiments demonstrate that subtle variations in RNA sequence might contribute to the high A-to-I editing levels found in humans.