Tasneem Islam

Avian Influenza H5-Containing Virus-Like Particles (VLPs): Host-Cell Receptor Specificity by STD NMR Spectroscopy†

H5N1 influenza virus is a highly contagious pathogen infecting poultry and other birds. The emergence of a human pandemic influenza virus from an avian progenitor appears to involve a switch in preferential binding of the influenza virus hemagglutinin (HA) from a(2,3)-linked N-acetylneuraminic acid containing glycans (the major form in the avian enteric tract) to a(2,6)-linked N-acetylneuraminic acid containing glycans (the major form in the human upper respiratory tract). Avian influenza viruses such as the H3 virus HA receptor typically contain the amino acids Gln226 and Gly228, which create a narrow binding pocket favoring a(2,3)-linked N-acetylneuraminic acid containing glycans. A number of other studies have also investigated HA–glycan recognition. Interestingly, the X-ray crystal structure of the reassembled HA of the 1918 Spanish influenza virus shows that its avianlike narrow binding pocket still allows highaffinity binding of a(2,6)-linked N-acetylneuraminic acid containing glycans. The mutation of only one amino acid (Asp190) within the HA binding site appears to determine the preference of the avianlike 1918 virus for human a(2,6)linked N-acetylneuraminic acid containing glycans. A mutation of Asp190 to Glu190 in the HA of the H5N1 strain could potentially switch its binding preference to a(2,6)linked glycans and consequently result in the emergence of a human pandemic virus. This fact emphasizes an urgent need to investigate the Nacetylneuraminic acid containing glycan recognition requirements by influenza virus HA that may lead to the development of novel antiinfluenza drugs that bind to the viral HA protein and consequently prevent the entry of the virus into human host cells. Rapid access to structural information would provide a detailed understanding of how virus particles interact with host cells on a molecular level and the determinants that prevent interspecies transmission of influenza viruses. Herein we present the first saturation transfer difference (STD) NMR spectroscopic study analyzing H5containing virus-like particles (VLPs) derived from the highly pathogenic avian H5N1 influenza strain in a complex with a(2,6)and a(2,3)-linked N-acetylneuraminides to mimic an in vitro or in vivo virus–host-cell interaction. We recently reported the production of H5 pseudotyped virus particles. To provide larger quantities of suitable VLPs for NMR studies, we have now successfully engineered heterologous H5 influenza VLPs by coexpression of pCDNA–synH5 coding for the hemagglutinin (H5) of H5N1 influenza virus and pCMV-dR8.91-expressing HIV-Gag-pol protein. Coexpression leads to spontaneous assembly of chimeric H5-VLPs, which contain the HA protein. The hemagglutinin, detected as uncleaved HA-0 precursor and HA-1/HA-2 cleaved mature forms, was incorporated on the surface of the viral particles at high levels. Expression of the viral HA protein was characterized by Western blot using specific C-terminal flag-tag M2 antibodies (Figure 1) and detection of VLPs by electron microscopy (Figure 2). In this study we investigated the capacity of H5-VLPs for competitive selection from a mixture of a(2,6)and a(2,3)sialyllactose (6’-SL and 3’-SL, respectively) of a preferred ligand (and therefore linkage) by means of STD NMR spectroscopy. It has been previously demonstrated that STD NMR spectroscopy can be utilized to investigate ligand interactions with whole virus particles, platelets, and intact cells. The large size of viruses and cells makes them attractive for studies with STD NMR spectroscopy because the inherently large line width enables saturation of the particle without affecting ligand signals. Additionally, the larger correlation time of bulky virus particles results in efficient spin diffusion and consequently stronger saturation transfer. To ensure the stability of the influenza H5-VLPs, NMR experiments were performed without prior purification, and 10% D2O was added for locking purposes. The H NMR spectrum of the influenza H5-VLPs (Figure 3a) shows the signals of the 20% sucrose cushion [*] Dr. T. Haselhorst, Dr. T. Islam, F. J. Rose, Prof. M. von Itzstein Institute for Glycomics, Griffith University, Gold Coast Campus Queensland, 4222 (Australia) Fax: (+61)7-555-29040 E-mail: m.vonitzstein@griffith.edu.au Homepage: http://www.griffith.edu.au/glycomics Dr. J.-M. Garcia, J. C. C. Lai, Prof. J. S. M. Peiris Hong Kong University Pasteur Research Centre Ltd., Dexter H.C. Man Building 8 Sassoon Road, Pokfulam, Hong Kong (China)

Design, synthesis and inhibitory activities of naringenin derivatives on human colon cancer cells

Based on the previous result, several naringenin derivatives modified at position 7 with bulky substituents were designed and synthesized, and their inhibitory effects on HCT116 human colon cancer cells were tested using a clonogenic assay. The half maximal inhibitory concentrations (IC(50)) of five naringenin derivatives ranged between 1.20 μM and 20.01 μM which are much better than naringenin used as a control. In addition, new structural modification at C-4 of flavanone results in improving both the anti-cancer effect and anti-oxidative effect. In vitro cyclin dependent kinase 2 (CDK2) binding assay was carried out based on the previous results. To elucidate the possible interaction between naringenin derivatives and CDK2, in silico docking study was performed. This result demonstrates the rationale for the different inhibitory activities of the naringenin derivatives. These findings could be used for designing cancer therapeutic or preventive flavanone-derived agents.

Further evidence that periodate cleavage of heparin occurs primarily through the antithrombin binding site

Porcine mucosal heparin was fragmented into low-molecular-weight (LMW) heparin by treatment of periodate-oxidized heparin with sodium hydroxide, followed by reduction with sodium borohydride and acid hydrolysis. Gradient polyacrylamide gel electrophoresis analysis showed a mixture of heparin fragments with an average size of eight disaccharide units. 1D 1H NMR showed two-thirds of the N-acetyl groups were lost on periodate cleavage, suggesting cleavage had occurred at the glucopyranosyluronic acid (GlcpA) and idopyranosyluronic acid (IdopA) residues located within and adjacent to the antithrombin III (ATIII) binding site. The N-acetyl glucopyranose (GlcpNAc) residue was lost on workup. The GlcpA residue, within the ATIII binding site, is on the non-reducing side of the N-sulfo, 3, 6-O-sulfo glycopyranosylamine (GlcpNS3S6S) residue. Thus, periodate cleaved heparin should be enriched in GlcpNS3S6S residues. Two-dimensional correlation spectroscopy (2D COSY) confirmed that LMW heparin prepared through periodate cleavage contained GlcpNS3S6S at its non-reducing end. As expected, this LMW heparin also showed reduced ATIII mediated anti-factor IIa and anti-factor Xa activities.

Unsaturated N-acetyl-D-glucosaminuronic acid glycosides as inhibitors of influenza virus sialidase

The threat of pandemic influenza is a significant concern of governments worldwide. There is a very limited and relatively expensive armament to tackle such a pandemic should it occur. This fact provides much impetus to the scientific community for the discovery of new and less expensive anti-influenza drugs. Our longstanding interest in the inhibition of influenza virus sialidase, coupled with the development of simple carbohydrates that mimic an unsaturated derivative of the enzymes naturally-occurring ligand, N-acetylneuraminic acid, has led us to investigate the development of influenza virus sialidase inhibitors based on these mimetics. We have successfully prepared a range of these compounds, in good yield, from the relatively inexpensive carbohydrate N-acetylglucosamine utilising a short synthetic procedure. We have employed a sialidase inhibition assay for biological evaluation of the target compounds and to our delight these mimetics have displayed significant inhibition of influenza virus sialidase.

C-4 Modified Sialosides Enhance Binding to Siglec-2 (CD22): Towards Potent Siglec Inhibitors for Immunoglycotherapy†

The regulatory functions of Siglecs (sialic acid binding immunoglobulin-like lectins) in the immune system provide opportunities for innovative therapeutic strategies for a wide range of immunological disorders or cancer (immunoglycotherapy). Siglec-2 (CD22), as a consequence of its pivotal role in B cell activation, has become an attractive target for therapies of autoimmune diseases and B cell-derived nonHodgkin s lymphoma (NHL). NHL is among the ten most common cancers with over 20000 deaths in 2010 for the US alone. Siglec-2 binds with high preference to a(2,6)-linked sialic acids (Sia), such as Neu5Aca(2,6)lactosamine (Scheme 1). Neu5Aca2Me (1) interacts with Siglec-2 mainly through 1) the negative charge on its carboxylate group, 2) the C-5 N-acetamido substituent, and 3) the glycerol side chain. Furthermore, replacement of the C-9 hydroxy group by an amino group did not interfere with binding to Siglecs. Crystallographic studies on Siglec-1 (sialoadhesin, Sn) demonstrated that acylation of this amino group enhances the overall affinity of the ligand for Siglecs by two to three orders of magnitude. The first breakthrough in the development of potent Siglec-2 inhibitors was the design of 9biphenylcarboxamido Neu5Aca2Me (9-BPC-Neu5Aca2Me, 2) which has a more than two orders of magnitude higher affinity to Siglec-2 than 1, and 2 has demonstrated potential to modulate signal transduction in B cells. Furthermore, based on 2, compounds were developed, which kill B cell lymphoma cells. Structural studies and modifications of the C-5 N-acyl substituent and the C-2 aglycon moiety of Nacetylneuraminic acid (Neu5Ac) have led to further improvement in affinity. Herein we report, for the first time the design, synthesis, and evaluation of a novel class of disubstituted Neu5Ac derivatives that is modified at the C-4 and C-9 positions of 1. Our structure-based design approach resulted in a promising novel lead compound 9-biphenylcarboxamido-4-m-nitrophenylcarboxamido-4,9-dideoxy Neu5Aca2Me (9-BPC-4mNPC-Neu5Aca2Me, 6b) that has sub-micromolar affinity for Siglec-2 and may provide a pathway for immunoglycotherapy strategies. An evaluation of our homology model (see Supporting Information) for Siglec-2 and other Siglecs led us to hypothesize that substituents at C-4 may provide additional interactions. To address this hypothesis we posed the following questions: 1) Can C-4 substituents enhance the interaction with Siglecs? 2) Do they interact specifically with the protein? 3) Do C-4 and C-9 modifications act synergistically? 4) Do the C-4 modified Neu5Ac derivatives bind to the same binding site as other Sia, such as 1?

Anti-Influenza Drug Discovery: Are We Ready for the Next Pandemic?

Publisher Summary This chapter examines the different aspects of the progress in anti-influenza drug discovery. Influenza viruses are enveloped single-stranded RNA viruses with a pleomorphic appearance and an average diameter of 120 nm. They are members of the orthomyxovirus family and can be classified into three serologically distinct types—A, B, and C. Both hemagglutinin and sialidase are carbohydrate-recognizing proteins and in humans recognize the well-known sialic acid, N-acetylneura minic acid, associated as the terminal carbohydrate unit of upper respiratory tract and lung-associated glycoconjugates. Influenza virus sialidase poses a highly attractive target for antiviral drug design due to its prominent position at the surface of the virion and its profound role in viral pathogenesis. The discovery of Zanamivir as a potent and selective inhibitor of influenza virus sialidase prompted several researchers to investigate the synthesis and structure–activity relationship studies of Neu5Ac2en-based compounds as potential sialidase inhibitors. Modifications of the guanidino function of Zanamivir at the primary nitrogen resulted in a dramatic reduction in the inhibition of neuraminidase (NA) and of viral replication in vivo. It is found that the success of such C-6 amides in the series against influenza A NA was in part due to a match between the spatial arrangement of the two binding pockets and the favored orientation of the amide rotamer that featured a trans-relationship between the larger substituent and the pyran ring.