Saturation transfer difference (STD) ¹H‐NMR experiments and in silico docking experiments to probe the binding of N‐acetylneuraminic acid and derivatives to Vibrio cholerae sialidase

Abstract

Saturation transfer difference (STD) (1)H NMR experiments were used to probe the epitope binding characteristics of the sialidase [EC 3.2.1.18] from the bacterium Vibrio cholerae, the causative agent of cholera. Binding preferences were investigated for N-acetylneuraminic acid (Neu5Ac, 1), the product of the sialidase catalytic reaction, for the known sialidase inhibitor 5-acetamido-2,6-anhydro-3,5-dideoxy-D-glycero-D-galacto-non-2-enoic acid (Neu5Ac2en, 2), and for the uronic acid-based Neu5Ac2en mimetic iso-propyl 2-acetamido-2,4-dideoxy-alpha-L-threo-hex-4-enopyranosiduronic acid (3), in which the native glycerol side-chain of Neu5Ac2en is replaced with an O-iso-propyl ether. The STD experiments provided evidence, supporting previous studies, that Neu5Ac (1) binds to the sialidase as the alpha-anomer. Docking experiments using DOCK (version 4.0.1) revealed further information regarding the binding characteristics of the enzyme active site in complex with Neu5Ac2en (2) and the Neu5Ac2en mimetic (3), indicating an expected dominant interaction of the acetamide moiety with the protein.

An investigation of the activity of recombinant rat skeletal muscle cytosolic sialidase

“…It is clear from this data that the cytosolic sialidase can accommodate hydrophobic groups in the C-6 glycerol binding domain. We have observed a similar hydrophobic group accommodation in microbial sialidases such as V. cholerae sialidase [37,41,42] and influenza virus sialidase [37]. Modest inhibition (18%) was also observed with the a2,3-linked substrate-like compound Neu5Ac-2-S-(a2,3)-Galb1Me 1, a thiosialoside metabolically stable to the action of sialidases [27].…”

A 1H STD NMR spectroscopic investigation of sialylnucleoside mimetics as probes of CMP-Kdn synthetase

“…We have had a longstanding interest [1][2][3] in the sialic acid family of carbohydrates, as well as sialic acid recognising proteins (SARPS), particularly enzymes, that are involved in their biosynthesis and degradation, and NMR spectroscopic methods for the investigation of enzymecatalysed reactions [4,5]. It is well known that nucleotide synthetases are essential for the biosynthesis of activated carbohydrates, commonly referred to as glycosyl donors.…”

“…The sialic acids (Sia), for example N-acetylneuraminic acid (Neu5Ac, 1), 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn, 2) and N-glycolylneuraminic acid (Neu5Gc, 3), are associated with a range of important glycoconjugates and the corresponding cytidine-5 -monophosphate sialic acid (CMP-Sia) is biosynthesised in the nucleus of eukaryotic cells, whereas all other nucleotide sugars are made in the cytoplasm. Our interest in sialic acids has led us to investigate the nucleotide synthetase that is essential for the activation of Kdn (2). Kdn (2), first discovered in fish egg polysialoglycoprotein [8], has been identified in Kdn-containing glycoconjugates from a large number of organisms ranging from bacteria to mammals [9][10][11].…”

CMP‐Pseudaminic Acid is a Natural Potent Inhibitor of PseB, the First Enzyme of the Pseudaminic Acid Pathway in Campylobacter jejuni and Helicobacter pylori

“…[14] STD NMR is rapid, easy to implement, does not require isotope labeling of the protein or excessive quantities of protein, and qualitative interpretation of the spectra is straightforward. [11,14,15] As an initial step towards the development of a small molecular inhibitor, binding epitopes were determined for PseB by monitoring the reaction directly in an NMR tube (13 8C) with proton and STD experiments at regular intervals ( Figure 2). For 1, the largest saturation transfer occurred at H1 of ribose indicating its close proximity to the protein.…”

“…Saturation transfer difference (STD) NMR is now widely used for studies examining the binding of carbohydrates to proteins. [11][12][13] STD NMR relies on the principle that when signals from a protein are selectively irradiated, ligands in exchange between the bound and the free forms will also become saturated. Subtraction of spectra acquired without protein irradiation reveals binding epitopes.…”