Paul David Madge

Differential carbohydrate binding and cell surface glycosylation of human cancer cell lines.

Currently there is only a modest level knowledge of the glycosylation status of immortalised cell lines that are commonly used in cancer biology as well as their binding affinities to different glycan structures. Through use of glycan and lectin microarray technology, this study has endeavoured to define the different bindings of cell surface carbohydrate structures to glycan‐binding lectins. The screening of breast cancer MDA‐MB435 cells, cervical cancer HeLa cells and colon cancer Caco‐2, HCT116 and HCT116–FM6 cells was conducted to determine their differential bindings to a variety of glycan and lectin structures printed on the array slides. An inverse relationship between the number of glycan structures recognised and the variety of cell surface glycosylation was observed. Of the cell lines tested, it was found that four bound to sialylated structures in initial screening. Secondary screening in the presence of a neuraminidase inhibitor (4‐deoxy‐4‐guanidino‐Neu5Ac2en) significantly reduced sialic acid binding. The array technology has proven to be useful in determining the glycosylation signatures of various cell‐lines as well as their glycan binding preferences. The findings of this study provide the groundwork for further investigation into the numerous glycan–lectin interactions that are exhibited by immortalised cell lines. J. Cell. Biochem. 112: 2230–2240, 2011.

Relative roles of GM1 ganglioside, N-acylneuraminic acids, and α2β1 integrin in mediating rotavirus infection.

ABSTRACT N-acetyl- and N-glycolylneuraminic acids (Sia) and α2β1 integrin are frequently used by rotaviruses as cellular receptors through recognition by virion spike protein VP4. The VP4 subunit VP8*, derived from Wa rotavirus, binds the internal N-acetylneuraminic acid on ganglioside GM1. Wa infection is increased by enhanced internal Sia access following terminal Sia removal from main glycan chains with sialidase. The GM1 ligand cholera toxin B (CTB) reduces Wa infectivity. Here, we found sialidase treatment increased cellular GM1 availability and the infectivity of several other human (including RV-3) and animal rotaviruses, typically rendering them susceptible to methyl α-d-N-acetylneuraminide treatment, but did not alter α2β1 usage. CTB reduced the infectivity of these viruses. Aceramido-GM1 inhibited Wa and RV-3 infectivity in untreated and sialidase-treated cells, and GM1 supplementation increased their infectivity, demonstrating the importance of GM1 for infection. Wa recognition of α2β1 and internal Sia were at least partially independent. Rotavirus usage of GM1 was mapped to VP4 using virus reassortants, and RV-3 VP8* bound aceramido-GM1 by saturation transfer difference nuclear magnetic resonance (STD NMR). Most rotaviruses recognizing terminal Sia did not use GM1, including RRV. RRV VP8* interacted minimally with aceramido-GM1 by STD NMR. Unusually, TFR-41 rotavirus infectivity depended upon terminal Sia and GM1. Competition of CTB, Sia, and/or aceramido-GM1 with cell binding by VP8* from representative rotaviruses showed that rotavirus Sia and GM1 preferences resulted from VP8*-cell binding. Our major finding is that infection by human rotaviruses of commonly occurring VP4 serotypes involves VP8* binding to cell surface GM1 glycan, typically including the internal N-acetylneuraminic acid. IMPORTANCE Rotaviruses, the major cause of severe infantile gastroenteritis, recognize cell surface receptors through virus spike protein VP4. Several animal rotaviruses are known to bind sialic acids at the termini of main carbohydrate chains. Conversely, only a single human rotavirus is known to bind sialic acid. Interestingly, VP4 of this rotavirus bound to sialic acid that forms a branch on the main carbohydrate chain of the GM1 ganglioside. Here, we use several techniques to demonstrate that other human rotaviruses exhibit similar GM1 usage properties. Furthermore, binding by VP4 to cell surface GM1, involving branched sialic acid recognition, is shown to facilitate infection. In contrast, most animal rotaviruses that bind terminal sialic acids did not utilize GM1 for VP4 cell binding or infection. These studies support a significant role for GM1 in mediating host cell invasion by human rotaviruses.

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?

A Secondary Sialic Acid Binding Site on Influenza Virus Neuraminidase: Fact or Fiction?

One flu over the cuckoos nest: The biological significance of a secondary sialic acid binding site on influenza virus neuraminidase remains elusive. On blocking the active site influenza-virus-containing virus-like particles with oseltamivir carboxylate, binding to a(2,3)-sialyllactose is still detected. Thus the sialyllactose must bind at a secondary sialic acid binding site (see structures: docking study of a(2,3)-sialyllactose in the secondary binding site of avian flu neuraminidase).

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.

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-β-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.