Neuraminidase inhibitor sensitivity and receptor-binding specificity of Cambodian clade 1 highly pathogenic H5N1 influenza virus.

The evolution of the highly pathogenic H5N1 influenza virus produces genetic variations that can lead to changes in antiviral susceptibility and in receptor-binding specificity. In countries where the highly pathogenic H5N1 virus is endemic or causes regular epidemics, the surveillance of these changes is important for assessing the pandemic risk. In Cambodia between 2004 and 2010, there have been 26 outbreaks of highly pathogenic H5N1 influenza virus in poultry and 10 reported human cases, 8 of which were fatal. We have observed naturally occurring mutations in hemagglutinin (HA) and neuraminidase (NA) of Cambodian H5N1 viruses that were predicted to alter sensitivity to neuraminidase inhibitors (NAIs) and/or receptor-binding specificity. We tested H5N1 viruses isolated from poultry and humans between 2004 and 2010 for sensitivity to the NAIs oseltamivir (Tamiflu) and zanamivir (Relenza). All viruses were sensitive to both inhibitors; however, we identified a virus with a mildly decreased sensitivity to zanamivir and have predicted that a V149A mutation is responsible. We also identified a virus with a hemagglutinin A134V mutation, present in a subpopulation amplified directly from a human sample. Using reverse genetics, we verified that this mutation is adaptative for human α2,6-linked sialidase receptors. The importance of an ongoing surveillance of H5N1 antigenic variance and genetic drift that may alter receptor binding and sensitivities of H5N1 viruses to NAIs cannot be underestimated while avian influenza remains a pandemic threat.

Design and synthesis of carbohydrate-based inhibitors of protein-carbohydrate interactions.

Our understanding of carbohydrate-protein interactions has significantly advanced over the past two years. In particular, a healthy amount of literature has appeared on selectins and their relevant ligands. A significant number of carbohydrate-metabolizing enzyme crystal structures have been solved which provide useful starting points for computer-assisted drug design. Some of these proteins have been implicated either directly or indirectly in playing roles in human-disease states, for example, in inflammation, in diabetes and its complications, and in microorganism-induced diseases such as influenza and cholera.

Targeting human respiratory syncytial virus transcription anti-termination factor M2-1 to inhibit in vivo viral replication.

Human respiratory syncytial virus (hRSV) is a leading cause of acute lower respiratory tract infection in infants, elderly and immunocompromised individuals. To date, no specific antiviral drug is available to treat or prevent this disease. Here, we report that the Smoothened receptor (Smo) antagonist cyclopamine acts as a potent and selective inhibitor of in vitro and in vivo hRSV replication. Cyclopamine inhibits hRSV through a novel, Smo-independent mechanism. It specifically impairs the function of the hRSV RNA-dependent RNA polymerase complex notably by reducing expression levels of the viral anti-termination factor M2-1. The relevance of these findings is corroborated by the demonstration that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine in vitro and that cyclopamine is able to reduce virus titers in a mouse model of hRSV infection. The results of our study open a novel avenue for the development of future therapies against hRSV infection.

Influenza virus sialidase: effect of calcium on steady-state kinetic parameters.

Ca2+ increases the initial rate of activity of sialidase from influenza virus (A/Tokyo/3/67). Increasing ionic strength also activates influenza virus sialidase. When ionic strength is controlled, smaller but still significant Ca2+ effects are observed, with Vmax/Km increased from 0.8.10(5) to 1.4.10(5) M-1 s-1 and Vmax increased from 6.3 to 9.5 s-1 by saturating Ca2+. The Ki of the competitive inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid was decreased from 2.7.10(-6) to 1.15.10(-6) M after the addition of saturating Ca2+. The data show that Ca2+ exerts a specific effect on Vmax/Km, leading to an increased rate of interaction of substrate with the enzyme. The Kd-app for the Ca2(+)-sialidase complex is 2 mM. Except for Mg2+ which behaves similarly to Ca2+, other mono- and divalent cations have little specific effect on sialidase kinetics. Sequence analysis of a range of subtypes of sialidases from influenza virus supports the proposal that Ca2+ binds at the subunit interface transmitting a conformational change to the enzyme active site. Ca2+ activation may have a physiological role in switching on sialidase activity during the release of newly synthesised virions from the host cell surface.

Analysis of inhibitor binding in influenza virus neuraminidase.

2,3-didehydro-2-deoxy-N:-acetylneuraminic acid (DANA) is a transition state analog inhibitor of influenza virus neuraminidase (NA). Replacement of the hydroxyl at the C9 position in DANA and 4-amino-DANA with an amine group, with the intention of taking advantage of an increased electrostatic interaction with a conserved acidic group in the active site to improve inhibitor binding, significantly reduces the inhibitor activity of both compounds. The three-dimensional X-ray structure of the complexes of these ligands and NA was obtained to 1.4 A resolution and showed that both ligands bind isosterically to DANA. Analysis of the geometry of the ammonium at the C4 position indicates that Glu119 may be neutral when these ligands bind. A computational analysis of the binding energies indicates that the substitution is successful in increasing the energy of interaction; however, the gains that are made are not sufficient to overcome the energy that is required to desolvate that part of the ligand that comes in contact with the protein.

Effect of substrate aglycon on enzyme mechanism in the reaction of sialidase from influenza virus.

The effect of substrate aglycon on enzyme mechanism of sialidase from influenza virus was investigated by kinetic isotope effects using the substrates 4-methylumbelliferyl-N-acetyl-alpha-D-neuraminic acid (Neu5Ac alpha 2MU) and p-nitrophenyl-N-acetyl-alpha-D-neuraminic acid (Neu5Ac alpha 2PNP). The kinetic isotope effect on Vmax (beta DV), at pH 6.0, as revealed by direct comparison of rates obtained with Neu5Ac alpha 2MU and the [3,3-2H]-substituted substrate analogue, was shown to be inverse. This indicates that sialidase-catalysed hydrolysis of Neu5Ac alpha 2MU proceeds with substantial positive charge development at the reaction centre in the transition state for the formation of the glycosyl cation-enzyme intermediate. However, no such inverse effect on Vmax at pH 6.0 was observed when using Neu5Ac alpha 2PNP and the [3,3-2H]-substituted substrate. A mechanism by which hydrolysis proceeds through an alpha-lactone intermediate has been proposed by Guo et al. [8]. We propose that the differences in beta DV for the substrates investigated are due primarily to the differing properties of the aglycon leaving groups, which may result in influenza virus sialidase catalysing substrate hydrolysis by a similar mechanism with alternative stabilisation of transition state.

Recent strategies in the search for new anti-influenza therapies.

Influenza is a highly contagious, acute upper respiratory tract disease caused by influenza virus, a member of the Orthomyxoviridae family. The viral particles have two surface antigens, haemagglutinin and sialidase (neuraminidase) that extensively decorate the surface of the virus and have been implicated in viral attachment and fusion, and the release of virion progeny, respectively. The receptor for haemagglutinin is the terminal sialic acid residue of host cell surface sialyloligosaccharides, while sialidase catalyses the hydrolysis of terminal sialic acid residues from sialyloligosaccharides. Extensive crystallographic studies of both these proteins have revealed that the residues that interact with the sialic acid are strictly conserved. Therefore, these proteins make attractive targets for the design of drugs to halt the progression of the virus. Recent successful efforts in the search for new cures for influenza have led to the development of three clinically-useful anti-influenza drugs. All three are potent, selective inhibitors of influenza virus A and B sialidase. Strategies for the development of haemagglutinin inhibitors have also been devised.

Molecular modeling studies on ligand binding to sialidase from influenza virus and the mechanism of catalysis.

A molecular modeling study has been used to investigate the structural and energetic aspects of substrate and inhibitor binding and the mechanism of catalysis of influenza virus sialidase. A detailed analysis of the interactions of both N-acetylneuraminic acid (Neu5Ac,1) and a number of transition-state analogues with the active site of influenza A sialidase at an atomic level is reported. In each case the calculated structures favorably agreed with the results from X-ray studies. A qualitative agreement between the calculated binding energies for inhibitors with positive substituents at the C4 position on the sugar ring and experimental Ki values was observed. We propose that the hydrolysis of sialosides occurs via an SN1 type mechanism that is facilitated through an activated solvent water molecule which can be expelled upon inhibitor binding. A reaction scheme is presented that is consistent with previously observed crystallographic structures, anomeric products, and isotope effects.

Synthesis and biological evaluation of N-acetylneuraminic acid-based rotavirus inhibitors.

Rotavirus can cause severe gastrointestinal disease, especially in infants and young children, and is particularly prevalent in Third-World countries. Therefore, the development of potential inhibitors of this virus is of great interest. The present study describes the synthesis and in vitro biological evaluation of a number of N-acetylneuraminic acid-based compounds as potential rotavirus inhibitors. Our data suggests that it is indeed possible to inhibit adhesion of the virus, and hence in vitro replication, with carbohydrate-based molecules, although this inhibition does appear to be strain dependent.

Synthesis and biological evaluation of sialylmimetics as rotavirus inhibitors.

Rotaviruses cause severe gastroenteritis in infants and are estimated to be responsible for over 600 000 deaths annually, primarily in developing countries. The development of potential inhibitors of this virus is therefore of great interest, particularly since the safety and efficacy of rotaviral vaccines has recently been questioned. This study describes the synthesis of a variety of compounds that can be considered as mimetics of N-acetylneuraminic acid thioglycosides and the subsequent in vitro biological evaluation of these sialylmimetics as inhibitors of rotaviral infection. Our results show that readily accessible carbohydrate-based compounds have the potential to act as inhibitors of rotaviral replication in vitro, presumably through inhibition of the rotaviral adhesion process.

A study of the active site of influenza virus sialidase: an approach to the rational design of novel anti-influenza drugs.

The development of sialidase inhibitor-based potential anti-influenza drugs using rational drug design techniques has been of recent interest. The present study details as investigation of the active site of influenza virus sialidase by using the program GRID in an attempt to design more potent inhibitors in the hope they will eventually lead to anti-influenza drugs. A number of different probes (amino, carboxy, hydroxy, methyl, etc) have been used in an effort to determine the functional groups most likely to improve the binding of the starting template 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en). The data have correctly predicted the binding regions for the carboxylate, acetamido (NH and methyl), and glycerol (OH) groups of N-acetylneuraminic acid. Moreover, the data suggest that the addition of certain functionalities (amino group) at the C-4 position should enhance the overall binding.

Investigation of the stability of thiosialosides toward hydrolysis by sialidases using NMR spectroscopy.

[formula: see text] 1H NMR spectroscopy has been used to investigate whether the alpha(2-->6)-linked thiosialoside 3 and the alpha(2-->3)-linked thiosialoside 9 are hydrolyzed in the presence of Vibrio cholerae sialidase. Similarly, the hydrolysis of the O-ketosides Neu5Ac-2-O-alpha-(2-->3)-Gal beta Me (4) and the alpha-(2-->6)-sialyllactoside 7, representing natural alpha(2-->3)- and alpha(2-->6)-linked sialosides, respectively, was investigated. The results of the 1H NMR experiments clearly demonstrate that the thiosialosides are not hydrolyzed by Vibrio cholerae sialidase. As expected, the O-sialosides are hydrolyzed to give N-acetyl-alpha-D-neuraminic acid as the first product of substrate cleavage.

Novel bismuth compounds have in vitro activity against Helicobacter pylori.

The increasing antimicrobial resistance in Helicobacter pylori has led to the search for new therapeutic agents. In this in vitro study, novel compounds combining bismuth with sialic acid inhibitors were investigated for bactericidal activity using time-kill methodology. The activity of these compounds was compared to bismuth subcitrate against a type strain and a clinical isolate of H. pylori. The compounds tested showed cidal activity which was related to the bismuth component of each drug. These compounds may offer a potential advantage over current bismuth preparations with the sialic acid inhibitor moiety interfering with adhesion of H. pylori to gastric epithelium.

A one-pot synthesis of novel N,N-dialkyl-S-glycosylsulfenamides.

An efficient new entry into N,N-dialkyl-S-glycosylsulfenamides is reported. The reaction of bis-activated alkyl halides in the presence of a secondary amine base with glycosylic S-acetyl derivatives (1-S-acetyl-1-thioaldoses or 2-S-acetyl-2-thioketoses) results in the formation of novel carbohydrate sulfonamides. These new carbohydrate-based sulfonamides may provide useful derivatives with biological activity, as well as provide reactive carbohydrate sulfonylating agents.

Rapid access to uronic acid-based mimetics of Kdn2en from D-glucurono-6,3-lactone.

A concise route to novel mimetics of Kdn2en, based on delta4-uronic acids, from D-glucurono-6,3-lactone is presented. Uronic acid-based mimetics in which an aliphatic ether (O-glycoside), a thioether (S-glycoside), or acetamide takes the place of the natural C-6 glycerol sidechain of the sialic acid were synthesized from the key intermediate, methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate bromide.

The synthesis and evaluation of novel sialic acid analogues bound to matrices for the purification of sialic acid-recognising proteins.

A novel N-acetylneuraminic acid analogue, 2-S-(5'-aminopentyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, as well as the thiosialoside 2-S-(2'-aminoethyl) 5-acetamido-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2- nonulopyranosidonic acid, have been synthesised and successfully coupled to CNBr-activated Sepharose 4B through the terminal amino group. The resultant affinity resins have proved efficient in purifying a number of sialic acid-recognising proteins such as Vibrio cholerae sialidase, sialidase-L from leech, trans-sialidase from Trypanosoma cruzi, and sialyltransferases from rat liver, all in high yield.

Synthesis of C-3 nitrogen-containing derivatives of N-acetyl-alpha,beta-D-mannosamine as substrates for N-acetylneuraminic acid aldolase.

The synthesis of 3-azido-3-deoxy, 3-amino-3-deoxy and 3-N-tert-butyloxycarbonyl-3-deoxy derivatives of 2-acetamido-2-deoxy-alpha,beta-D-mannose (N-acetyl-alpha,beta-D-mannosamine, ManNAc), is presented. The 3-azido-3-deoxy- and 3-N-tert-butyloxycarbonyl compounds were further characterised as their peracetates. A preliminary study has found that these C-3 nitrogen-substituted derivatives of ManNAc not to be substrates for Neu5Ac aldolase.