RESUMEN
We report the structural and functional characterization of a novel heparanase (BpHep) from the invasive pathogenic bacterium Burkholderia pseudomallei (Bp), showing â¼24% sequence identity with human heparanase (hHep). Site-directed mutagenesis studies confirmed the active site resi-dues essential for activity, and we found that BpHep has specificity for heparan sulfate. Finally, we describe the first heparanase X-ray crystal structure, which provides new insight into both substrate recognition and inhibitor design.
Asunto(s)
Burkholderia pseudomallei/enzimología , Glucuronidasa/química , Glucuronidasa/metabolismo , Cristalografía por Rayos X , Glucuronidasa/aislamiento & purificación , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
A new direction for influenza virus sialidase inhibitor development was identified using a sulfonate congener of 2-deoxy-2-ß-H N-acetylneuraminic acid. Sialosyl sulfonates can be synthesised efficiently in four steps from N-acetylneuraminic acid via a microwave assisted decarboxylation. The presence of the sulfonate group significantly increases inhibition of influenza virus sialidase and viral infection when compared to the carboxylate congener, and also to the benchmark sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, Neu5Ac2en.
Asunto(s)
Antivirales/farmacología , Arilsulfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/efectos de los fármacos , Neuraminidasa/antagonistas & inhibidores , Replicación Viral/efectos de los fármacos , Antivirales/síntesis química , Antivirales/química , Arilsulfonatos/síntesis química , Arilsulfonatos/química , Conformación de Carbohidratos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Neuraminidasa/metabolismo , Relación Estructura-ActividadRESUMEN
Novel 3,4-disubstituted-Neu5Ac2en derivatives have been synthesised to probe the open 150-loop conformation of influenza virus sialidases. Both equatorially and axially (epi) substituted C4 amino and guanidino 3-(p-tolyl)allyl-Neu5Ac2en derivatives were prepared, via the 4-epi-hydroxy derivative. The equatorially-substituted 4-amino derivative showed low micromolar inhibition of both group-1 (pdm09 H1N1) and group-2 (pdm57 H2N2) sialidases, and provides the first in vitro evidence that a group-2 sialidase may exhibit 150-loop flexibility.
Asunto(s)
Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidasa/metabolismo , Orthomyxoviridae/enzimología , Ácidos Siálicos/química , Sondas Moleculares , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/química , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
The binding of a natural anthocyanin to influenza neuraminidase has been studied employing mass spectrometry and molecular docking. Derived from a black elderberry extract, cyanidin-3-sambubiocide has been found to be a potent inhibitor of sialidase activity. This study reveals the molecular basis for its activity for the first time. The anthocyanin is shown by parallel experimental and computational approaches to bind in the so-called 430-cavity in the vicinity of neuraminidase residues 356-364 and 395-432. Since this antiviral compound binds remote from Asp 151 and Glu 119, two residues known to regulate neuraminidase resistance, it provides the potential for the development of a new class of antivirals against the influenza virus without this susceptibility.
Asunto(s)
Antocianinas/antagonistas & inhibidores , Disacáridos/química , Virus de la Influenza A/enzimología , Neuraminidasa/metabolismo , Antocianinas/química , Electroforesis en Gel de Agar , Espectrometría de Masas , Estructura Molecular , Neuraminidasa/química , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Mitochondrial complex II (CII) has been recently identified as a novel target for anti-cancer drugs. Mitochondrially targeted vitamin E succinate (MitoVES) is modified so that it is preferentially localized to mitochondria, greatly enhancing its pro-apoptotic and anti-cancer activity. Using genetically manipulated cells, MitoVES caused apoptosis and generation of reactive oxygen species (ROS) in CII-proficient malignant cells but not their CII-dysfunctional counterparts. MitoVES inhibited the succinate dehydrogenase (SDH) activity of CII with IC(50) of 80 µM, whereas the electron transfer from CII to CIII was inhibited with IC(50) of 1.5 µM. The agent had no effect either on the enzymatic activity of CI or on electron transfer from CI to CIII. Over 24 h, MitoVES caused stabilization of the oxygen-dependent destruction domain of HIF1α fused to GFP, indicating promotion of the state of pseudohypoxia. Molecular modeling predicted the succinyl group anchored into the proximal CII ubiquinone (UbQ)-binding site and successively reduced interaction energies for serially shorter phytyl chain homologs of MitoVES correlated with their lower effects on apoptosis induction, ROS generation, and SDH activity. Mutation of the UbQ-binding Ser(68) within the proximal site of the CII SDHC subunit (S68A or S68L) suppressed both ROS generation and apoptosis induction by MitoVES. In vivo studies indicated that MitoVES also acts by causing pseudohypoxia in the context of tumor suppression. We propose that mitochondrial targeting of VES with an 11-carbon chain localizes the agent into an ideal position across the interface of the mitochondrial inner membrane and matrix, optimizing its biological effects as an anti-cancer drug.
Asunto(s)
Antineoplásicos/administración & dosificación , Apoptosis/efectos de los fármacos , Sistemas de Liberación de Medicamentos/métodos , Complejo II de Transporte de Electrones/metabolismo , Mitocondrias/metabolismo , Vitamina E/administración & dosificación , Animales , Antineoplásicos/farmacología , Bovinos , Transporte de Electrón , Humanos , Concentración 50 Inhibidora , Células Jurkat , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa , Vitamina E/farmacologíaRESUMEN
Novel 3-C-alkylated-Neu5Ac2en derivatives have been designed to target the expanded active site cavity of influenza virus sialidases with an open 150-loop, currently seen in X-ray crystal structures of influenza A virus group-1 (N1, N4, N5, N8), but not group-2 (N2, N9), sialidases. The compounds show selectivity for inhibition of H5N1 and pdm09 H1N1 sialidases over an N2 sialidase, providing evidence of the relative 150-loop flexibility of these sialidases. In a complex with N8 sialidase, the C3 substituent of 3-phenylally-Neu5Ac2en occupies the 150-cavity while the central ring and the remaining substituents bind the active site as seen for the unsubstituted template. This new class of inhibitors, which can 'trap' the open 150-loop form of the sialidase, should prove useful as probes of 150-loop flexibility.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Virus de la Influenza A/enzimología , Ácido N-Acetilneuramínico/análogos & derivados , Neuraminidasa/antagonistas & inhibidores , Alquilación , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Modelos Moleculares , Estructura Molecular , Ácido N-Acetilneuramínico/síntesis química , Ácido N-Acetilneuramínico/química , Ácido N-Acetilneuramínico/farmacología , Neuraminidasa/metabolismo , Docilidad/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
We used NMR spectroscopy, molecular modeling and infectivity competition assays to investigate the key interactions between the spike protein (VP8(*)) from 'sialidase-insensitive' human Wa and 'sialidase-sensitive' porcine CRW-8 rotaviruses and the glycans of gangliosides G(M1) and G(D1a). Our data provide strong evidence that N-acetylneuraminic acid is a key determinant for binding of these rotaviruses. This is in contrast to the widely accepted paradigm that sialic acids are irrelevant in host cell recognition by sialidase-insensitive rotaviruses.
Asunto(s)
Ácido N-Acetilneuramínico/metabolismo , Rotavirus/patogenicidad , Espectroscopía de Resonancia Magnética , Rotavirus/metabolismoRESUMEN
Influenza virus infection continues to cause significant, often severe, respiratory illness worldwide. A validated target for the development of anti-influenza agents is the virus surface protein sialidase. In the current study, we have discovered a highly potent inhibitor of influenza virus sialidase, based on a novel sialosyl sulfonate template. The synthesised 3-guanidino sialosyl α-sulfonate, a sulfonozanamivir analogue, inhibits viral replication inâ vitro at the nanomolar level, comparable to that of the anti-influenza drug zanamivir. Using protein X-ray crystallography we show that the sialosyl α-sulfonate template binds within the sialidase active site in a 1 C4 chair conformation. The C1-sulfonate moiety forms crucial and strong-binding interactions with the active site's triarginyl cluster, while the 3-guanidino moiety interacts significantly with conserved active site residues. This sulfonozanamivir analogue provides a new direction in anti-influenza virus drug development.
Asunto(s)
Antivirales/química , Antivirales/farmacología , Arilsulfonatos/química , Arilsulfonatos/farmacología , Virus de la Influenza A/efectos de los fármacos , Zanamivir/análogos & derivados , Zanamivir/farmacología , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Virus de la Influenza A/química , Virus de la Influenza A/enzimología , Virus de la Influenza A/fisiología , Gripe Humana/tratamiento farmacológico , Gripe Humana/virología , Simulación del Acoplamiento Molecular , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/química , Neuraminidasa/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Mitochondria have recently emerged as new and promising targets for cancer prevention and therapy. One of the reasons for this is that mitochondria are instrumental to many types of cell death and often lie downstream from the initial actions of anti-cancer drugs. Unlike the tumour suppressor gene encoding p53 that is notoriously prone to inactivating mutations but whose function is essential for induction of apoptosis by DNA-targeting agents (such as doxorubicin or 5-fluorouracil), mitochondria present targets that are not so compromised by genetic mutation and whose targeting overcomes problems with mutations of upstream targets such as p53. We have recently proposed a novel class of anti-cancer agents, mitocans that exert their anti-cancer activity by destabilising mitochondria, promoting the selective induction of apoptotic death in tumour cells. In this communication, we review recent findings on mitocans and propose a common basis for their mode of action in inducing apoptosis of cancer cells. We use as an example the analogues of vitamin E that are proving to be cancer cell-specific and may soon be developed into efficient anti-cancer drugs.
Asunto(s)
Antineoplásicos/farmacología , Mitocondrias/efectos de los fármacos , Vitamina E/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacosAsunto(s)
Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/química , Orthomyxoviridae/enzimología , Animales , Antivirales/farmacología , Sitios de Unión/efectos de los fármacos , Humanos , Modelos Moleculares , Neuraminidasa/metabolismo , Resonancia Magnética Nuclear Biomolecular , Orthomyxoviridae/química , Infecciones por Orthomyxoviridae/enzimología , Oseltamivir/farmacologíaAsunto(s)
Gangliósido G(M3)/metabolismo , Rotavirus/química , Rotavirus/metabolismo , Animales , Secuencia de Carbohidratos , Gangliósido G(M3)/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Relación Estructura-Actividad , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismoRESUMEN
Human parainfluenza viruses (hPIVs) cause upper and lower respiratory tract disease in children that results in a significant number of hospitalizations and impacts health systems worldwide. To date, neither antiviral drugs nor vaccines are approved for clinical use against parainfluenza virus, which reinforces the urgent need for new therapeutic discovery strategies. Here we use a multidisciplinary approach to develop potent inhibitors that target a structural feature within the hPIV type 3 haemagglutinin-neuraminidase (hPIV-3 HN). These dual-acting designer inhibitors represent the most potent designer compounds and efficiently block both hPIV cell entry and virion progeny release. We also define the binding mode of these inhibitors in the presence of whole-inactivated hPIV and recombinantly expressed hPIV-3 HN by Saturation Transfer Difference NMR spectroscopy. Collectively, our study provides an antiviral preclinical candidate and a new direction towards the discovery of potential anti-parainfluenza drugs.
Asunto(s)
Antivirales/química , Diseño de Fármacos , Hemaglutininas Virales/química , Neuraminidasa/antagonistas & inhibidores , Virus de la Parainfluenza 3 Humana , Dominio Catalítico , Línea Celular Tumoral , Simulación por Computador , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Humanos , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Conformación Molecular , Simulación de Dinámica Molecular , Ácidos Neuramínicos/química , Proteínas Recombinantes/química , Solventes/química , Propiedades de SuperficieRESUMEN
Human parainfluenza virus type 1 is the major cause of croup in infants and young children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid (Neu5Ac2en)-based inhibitor development. In this study, we explore the effect of C-5 modifications on the potency of Neu5Ac2en derivatives that target the human parainfluenza type-1 hemagglutinin-neuraminidase protein. Our study demonstrates that the replacement of the Neu5Ac2en C-5 acetamido moiety with more hydrophobic alkane-based moieties improves the inhibitory potency for both hemagglutinin-neuraminidase functions. These findings shed light on the importance of C-5 substitution on Neu5Ac2en in the design of novel sialic acid-based inhibitors that target human parainfluenza type-1 hemagglutinin-neuraminidase.
Asunto(s)
Amidas/farmacología , Antivirales/farmacología , Proteína HN/metabolismo , Virus de la Parainfluenza 1 Humana/efectos de los fármacos , Amidas/química , Animales , Antivirales/química , Línea Celular , Evaluación Preclínica de Medicamentos , Proteína HN/química , Concentración 50 Inhibidora , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
A series of C3 O-functionalized 2-acetamido-2-deoxy-Δ4-ß-D-glucuronides were synthesized to explore noncharge interactions in subsite 2 of the influenza virus sialidase active site. In complex with A/N8 sialidase, the parent compound (C3 OH) inverts its solution conformation to bind with all substituents well positioned in the active site. The parent compound inhibits influenza virus sialidase at a sub-µM level; the introduction of small alkyl substituents or an acetyl group at C3 is also tolerated.
Asunto(s)
Acetamidas/química , Antivirales/química , Glucurónidos/química , Neuraminidasa/química , Orthomyxoviridae/enzimología , Acetamidas/síntesis química , Dominio Catalítico , Pruebas de Enzimas , Fluorometría , Glucurónidos/síntesis química , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Neuraminidasa/antagonistas & inhibidores , Electricidad Estática , Relación Estructura-ActividadRESUMEN
SCOPE: Vitamin E (VE) analogues, epitomised by the prototypic α-tocopheryl succinate (α-TOS), are potent anti-cancer agents. α-TOS has recently been shown to trigger apoptosis by targeting the ubiquinone (UbQ) binding site(s) of the mitochondrial complex II (CII) and to cause excessive production of reactive oxygen species. METHODS AND RESULTS: We have modelled, using two approaches, a range of VE analogues into the proximal UbQ (Q(p) ) binding site of CII. This study reveals that in both cases, the calculated interaction energies of individual VE analogues correlate (R(2) value >0.8) with their toxicity to cancer cells. CONCLUSION: These data further support the UbQ site(s) of CII as an important target determining the anti-cancer activity of VE analogues as well as other emerging anti-cancer drugs.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Complejo II de Transporte de Electrones/metabolismo , Vitamina E/análogos & derivados , Sitios de Unión , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Relación Estructura-Actividad , Ubiquinona/metabolismoRESUMEN
Rotaviruses ubiquitously infect children under the age of 5, being responsible for more than half a million diarrhoeal deaths each year worldwide. Host cell oligosaccharides containing sialic acid(s) are critical for attachment by rotaviruses. However, to date, no detailed three-dimensional atomic model showing the exact rotavirus interactions with these glycoconjugate receptors has been reported. Here, we present the first crystallographic structures of the rotavirus carbohydrate-recognizing protein VP8* in complex with ganglioside G(M3) glycans. In combination with assessment of the inhibition of rotavirus infectivity by N-acetyl and N-glycolyl forms of this ganglioside, our results reveal key details of rotavirus-ganglioside G(M3) glycan recognition. In addition, they show a direct correlation between the carbohydrate specificities exhibited by VP8* from porcine and by monkey rotaviruses and the respective infectious virus particles. These novel results also indicate the potential binding interactions of rotavirus VP8* with other sialic acid-containing gangliosides.
Asunto(s)
Gangliósidos/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Polisacáridos/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Infecciones por Rotavirus/metabolismo , Rotavirus/patogenicidad , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Animales , Cristalografía por Rayos X , Haplorrinos , Espectroscopía de Resonancia Magnética , Modelos Químicos , Conformación Proteica , Infecciones por Rotavirus/virología , PorcinosRESUMEN
Influenza virus sialidase has an essential role in the virus' life cycle. Two distinct groups of influenza A virus sialidases have been established, that differ in the flexibility of the '150-loop', providing a more open active site in the apo form of the group-1 compared to group-2 enzymes. In this study we show, through a multidisciplinary approach, that novel sialic acid-based derivatives can exploit this structural difference and selectively inhibit the activity of group-1 sialidases. We also demonstrate that group-1 sialidases from drug-resistant mutant influenza viruses are sensitive to these designed compounds. Moreover, we have determined, by protein X-ray crystallography, that these inhibitors lock open the group-1 sialidase flexible 150-loop, in agreement with our molecular modelling prediction. This is the first direct proof that compounds may be developed to selectively target the pandemic A/H1N1, avian A/H5N1 and other group-1 sialidase-containing viruses, based on an open 150-loop conformation of the enzyme.
RESUMEN
With the global spread of the pandemic H1N1 and the ongoing pandemic potential of the H5N1 subtype, the influenza virus represents one of the most alarming viruses spreading worldwide. The influenza virus sialidase is an effective drug target, and a number of inhibitors are clinically effective against the virus (zanamivir, oseltamivir, peramivir). Here we report structural and biochemical studies of the human cytosolic sialidase Neu2 with influenza virus sialidase-targeting drugs and related compounds.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Neuraminidasa/antagonistas & inhibidores , Orthomyxoviridae/enzimología , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Subtipo H5N1 del Virus de la Influenza A/enzimología , Modelos Moleculares , Conformación Molecular , Neuraminidasa/química , Neuraminidasa/metabolismoRESUMEN
Influenza virus remains a significant threat to humanity despite the discovery of novel anti-viral therapies and the continuing development of seasonal vaccines. The reason for this ongoing concern is that the development of drug resistance to anti-virals has rapidly occurred and the currently developed vaccines are typically only effective against a specific influenza virus strain. The continual emergence of new influenza virus strains that may lead to the next human pandemic has inspired much research into a better understanding of the virus, particularly the role(s) of carbohydrates in the virus' lifecycle. Much of this research is directed towards next generation anti-influenza drugs. Important advances in the interrogation of influenza virus' surface glycoprotein haemagglutinin by NMR spectroscopy have been made in recent times. An overview of some of these advances is provided.
Asunto(s)
Antivirales/farmacología , Subtipo H5N1 del Virus de la Influenza A/efectos de los fármacos , Gripe Humana/tratamiento farmacológico , Metabolismo de los Hidratos de Carbono , Diseño de Fármacos , Farmacorresistencia Viral , Hemaglutininas Virales/metabolismo , Humanos , Gripe Humana/epidemiología , Gripe Humana/virología , Espectroscopía de Resonancia MagnéticaRESUMEN
Both, the influenza C (INF-C) virus haemagglutinin esterase fusion and bovine coronavirus (BCoV) haemagglutinin esterase surface glycoproteins exhibit a lectin binding capability and a receptor-destroying 9-O-acetyl esterase activity that recognise 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2))-containing glycans. Here we report nuclear magnetic resonance and molecular modelling studies on the 9-O-acetyl esterase showing that the alpha-configured Neu5,9Ac(2) is strictly preferred by the INF-C and BCoV esterases. Interestingly, we have discovered that the INF-C esterase function releases acetate independently of the chemical nature of the aglycon moiety, whereas subtle differences in substrate recognition were found for BCoV esterase. Analysis of the apo and complexed X-ray crystal structure of INF-C esterase revealed that binding of 9-O-acetylated N-acetylneuraminic acids is a dynamic process that involves conformational rearrangement of serine-57 in the esterase active site. This study provides valuable insights towards the design of drugs to combat INF-C virus and coronavirus infections causing outbreaks of upper respiratory infections and severe diarrhea in calves, respectively.