Efficient Blocking of Enterovirus 71 Infection by Heparan Sulfate Analogues Acting as Decoy Receptors.

Enterovirus 71 (EV71) is a major etiological agent of hand, foot, and mouth disease, for which there is no antiviral therapy. We have developed densely sulfated disaccharide heparan sulfate (HS) analogues that are potent small molecule inhibitors of EV71 infection, binding to the viral capsid and acting as decoy receptors to block early events of virus replication. The simplified structures, more potent than defined HS disaccharides and with no significant anticoagulant activity, offer promise as anti-EV71 agents.

New antiviral approaches for human parainfluenza: Inhibiting the haemagglutinin-neuraminidase.

Human parainfluenza viruses cause acute respiratory tract infections and disease predominantly in young children and immunocompromised individuals. Currently, there are no vaccines to prevent hPIV infections, nor licensed anti-hPIV drugs. There is therefore a need for specific antiviral therapies to decrease the morbidity and mortality associated with hPIV diseases. Haemagglutinin-neuraminidase (HN) is one of two hPIV surface proteins with critical roles in host receptor recognition, binding and cleavage; it has been explored as a key drug development target for the past few decades with variable success. Recent advancements in computational modelling and the availability of the X-ray crystal structure of hPIV3 HN have improved our understanding of the structural and mechanistic features of HN. This review explores structural features of the HN protein that are being exploited for structure-guided inhibitor design. We describe past and present hPIV HN inhibition strategies based on sialic acid scaffolds, together with other novel approaches that decrease hPIV infectivity. Although many HN inhibitors have been developed and evaluated as anti-hPIV agents, currently only a host-directed therapy (DAS181) has succeeded in phase II clinical drug trials. Hence, the review concludes with future considerations for targeting the specific function(s) of hPIV HN and suggestions for antiviral drug design.

Structural Insights into Human Parainfluenza Virus 3 Hemagglutinin-Neuraminidase Using Unsaturated 3- N-Substituted Sialic Acids as Probes.

A novel approach to human parainfluenza virus 3 (hPIV-3) inhibitor design has been evaluated by targeting an unexplored pocket within the active site region of the hemagglutinin-neuraminidase (HN) of the virus that is normally occluded upon ligand engagement. To explore this opportunity, we developed a highly efficient route to introduce nitrogen-based functionalities at the naturally unsubstituted C-3 position on the neuraminidase inhibitor template N-acyl-2,3-dehydro-2-deoxy-neuraminic acid ( N-acyl-Neu2en), via a regioselective 2,3-bromoazidation. Introduction of triazole substituents at C-3 on this template provided compounds with low micromolar inhibition of hPIV-3 HN neuraminidase activity, with the most potent having 48-fold improved potency over the corresponding C-3 unsubstituted analogue. However, the C-3-triazole N-acyl-Neu2en derivatives were significantly less active against the hemagglutinin function of the virus, with high micromolar IC50 values determined, and showed insignificant in vitro antiviral activity. Given the different pH optima of the HN protein's neuraminidase (acidic pH) and hemagglutinin (neutral pH) functions, the influence of pH on inhibitor binding was examined using X-ray crystallography and STD NMR spectroscopy, providing novel insights into the multifunctionality of hPIV-3 HN. While the 3-phenyltriazole- N-isobutyryl-Neu2en derivative could bind HN at pH 4.6, suitable for neuraminidase inhibition, at neutral pH binding of the inhibitor was substantially reduced. Importantly, this study clearly demonstrates for the first time that potent inhibition of HN neuraminidase activity is not necessarily directly correlated with a strong antiviral activity, and suggests that strong inhibition of the hemagglutinin function of hPIV HN is crucial for potent antiviral activity. This highlights the importance of designing hPIV inhibitors that primarily target the receptor-binding function of hPIV HN.

A Sulfonozanamivir Analogue Has Potent Anti-influenza Virus Activity.

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 in 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.

A sialosyl sulfonate as a potent inhibitor of influenza virus replication.

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.

Structural characterisation of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma (BL) Daudi cells by NMR spectroscopy.

Siglec-2 undergoes constitutive endocytosis and is a drug target for autoimmune diseases and B cell-derived malignancies, including hairy cell leukaemia, marginal zone lymphoma, chronic lymphocytic leukaemia and non-Hodgkin's lymphoma (NHL). An alternative to current antibody-based therapies is the use of liposomal nanoparticles loaded with cytotoxic drugs and decorated with Siglec-2 ligands. We have recently designed the first Siglec-2 ligands (9-biphenylcarboxamido-4-meta-nitrophenyl-carboxamido-Neu5Acα2Me, 9-BPC-4-mNPC-Neu5Acα2Me) with simultaneous modifications at C-4 and C-9 position. In the current study we have used Saturation Transfer Difference (STD) NMR spectroscopy to monitor the binding of 9-BPC-4-mNPC-Neu5Acα2Me to Siglec-2 present on intact Burkitt's lymphoma Daudi cells. Pre-treatment of cells with periodate resulted in significantly higher STD NMR signal intensities for 9-BPC-4-mNPC-Neu5Acα2Me as the cells were more susceptible to ligand binding because cis-binding on the cell surface was removed. Quantification of STD NMR effects led to a cell-derived binding epitope of 9-BPC-4-mNPC-Neu5Acα2Me that facilitated the design and synthesis of C-2, C-3, C-4 and C-9 tetra-substituted Siglec-2 ligands showing an 88-fold higher affinity compared to 9-BPC-Neu5Acα2Me. This is the first time a NMR-based binding study of high affinity Siglec-2 (CD22) ligands in complex with whole Burkitt's lymphoma Daudi cells has been described that might open new avenues in developing tailored therapeutics and personalised medicine.

Functional and structural characterization of a heparanase.

We report the structural and functional characterization of a novel heparanase (BpHep) from the invasive pathogenic bacterium Burkholderia pseudomallei (Bp), showing ∼24% 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.

Access to 3-O-Functionalized N-Acetylneuraminic Acid Scaffolds.

Direct access to 3-O-functionalized 2-α-N-acetylneuraminides and their corresponding 2,3-dehydro-2-deoxy-N-acetylneuraminic acid derivatives is described. Initially, a stereoselective ring-opening of the key intermediate N-acetylneuraminic acid (Neu5Ac) 2,3-ß-epoxide with an alcohol provided the 3-hydroxy α-glycoside. O-Alkylation of the C3 hydroxyl group generated novel 3-O-functionalized Neu5Ac derivatives that provided the corresponding unsaturated derivatives upon elimination.

Novel 3,4-disubstituted-Neu5Ac2en derivatives as probes to investigate flexibility of the influenza virus sialidase 150-loop.

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.

Uronosyl phosphonate-based sialidase inhibitor synthesis and conformational analysis.

With a view to development of novel sialidase inhibitors, mimetics of the natural inhibitor Neu5Ac2en have been prepared in which a phosphonate group replaces the sialic acid glycerol side chain. Different hex-4-en derivatives adopt half-chair conformations that place the glycosyl phosphonate in an equatorial position. For the α-L-threo-hex-4-en derivative this conformation is equivalent to that of Neu5Ac2en, and opposite to that seen for alkyl O-glycosides with the same overall stereochemistry.

Exploring the interactions of unsaturated glucuronides with influenza virus sialidase.

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.

Synthesis and evaluation of novel 3-C-alkylated-Neu5Ac2en derivatives as probes of influenza virus sialidase 150-loop flexibility.

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.

Accessing C-1 phosphonylated 2-acylamino uronic acids via 2-nitro-glycals.

Two approaches are described for the synthesis of 2-acylamino uronic acid glycosyl phosphonates from readily accessible D-glucal. The first approach that entailed oxidation of the C-6 hydroxyl group followed by phosphonylation of the uronate 2-nitro-glycal, resulted in the formation of the ß-L-gulo-configured phosphonate. Reversing the reaction order resulted in the exclusive formation of the ß-D-gluco-configured phosphonate. In both cases the thermodynamic 1,2-trans-di-equatorial phosphonylation product is obtained.

Novel sialic acid derivatives lock open the 150-loop of an influenza A virus group-1 sialidase.

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.

Complexity in influenza virus targeted drug design: interaction with human sialidases.

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.

Cracking the code for H5N1-bird flu and beyond.

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.

Synthesis and biological evaluation of galactofuranosyl alkyl thioglycosides as inhibitors of mycobacteria.

As part of our research interest directed toward the development of antimycobacterial agents, we have investigated compounds based on galactofuranose (Galf), an essential cell wall component of mycobacteria. The objective of this study was to explore structure activity relationships of Galf thioglycosides with straight chain and branched aglycons. Acylated Galf 9-heptadecyl thioglycoside was prepared by Lewis acid-catalyzed thioglycosidation of 1,2,3,5,6-penta-O-acyl-D-galactofuranose with 9-heptadecanethiol, and subsequently converted to the corresponding sulfone using m-CPBA. Both Galf 9-heptadecyl thioglycoside and sulfone displayed in vitro inhibition (MIC) of the growth of Mycobacterium smegmatis below 5 microg/mL, while Galf 1-octyl thioglycoside gave no inhibition at or below 32 microg/mL.

Synthesis and evaluation of galactofuranosyl N,N-dialkyl sulfenamides and sulfonamides as antimycobacterial agents.

The recent emergence of clinically oppressive superbugs, some with resistance to nearly all frontline drug therapies, has challenged our ability to combat such infectious organisms as Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Our medicinal chemistry program targeting this pathogen has identified several potent galactofuranose-based in vitro inhibitors of mycobacterial growth. The most potent compound, the Galf N,N-didecyl sulfenamide 8d, displayed anti-mycobacterial activity (MIC) of 1 microg/mL in a cell based assay against a representative strain of Mycobacterium smegmatis.

Synthesis and evaluation of 4-O-alkylated 2-deoxy-2,3-didehydro-N-acetylneuraminic acid derivatives as inhibitors of human parainfluenza virus type-3 sialidase activity.

The X-ray crystal structure of the paramyxoviral surface glycoprotein haemagglutinin-neuraminidase (HN) from Newcastle Disease virus was used as a template to design inhibitors of the HN from human parainfluenza virus type-3 (hPIV-3). 4-O-Alkylated derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en), accessed from 8,9-O-isopropylidenated-Neu5Ac2en1Me, were found to inhibit the sialidase (neuraminidase) activity of hPIV-3 (strain C243) in the range of 3-30muM. This is comparable or improved activity compared to the parent 4-hydroxy compound.