This review focuses on the application of NMR methods for understanding, at the molecular and atomic levels, the diverse mechanisms by which sugar molecules are recognised by the binding sites of lectins, antibodies and enzymes. Given the intrinsic chemical natures of sugars and their flexibility, it is well established that NMR parameters should be complemented by computational methods in attempts to unravel the structural and conformational features of the molecular recognition process unambiguously. We therefore aim here to describe new and significant advances in the knowledge of carbohydrate-protein interactions, obtained by employing state-of-the-art NMR and molecular modelling. We have not attempted to prepare an exhaustive review but have tried to focus on describing the key aspects that should be considered when tackling a problem within this research topic.
Herein, we describe the use of thioglycosides as glycosidase inhibitors by employing novel modifications at the reducing end of these glycomimetics. The inhibitors display a basic galactopyranosyl unit (1→4)-bonded to a 3-deoxy-4-thiopentopyranose moiety. The molecular basis of the observed inhibition has been studied by using a combination of NMR spectroscopy and molecular modeling techniques. It is demonstrated that these molecules are not recognized by Escherichia coli β-galactosidase in their ground-state conformation, with a conformational selection process taking place. In fact, the observed conformational distortion depends on the chemical nature of the compounds and results from the rotation around the glycosidic linkage (variation of Φ or Ψ) or from the deformation of the six-membered ring of the pentopyranose. The bound conformations of the ligand are adapted in the enzymatic pocket with a variety of hydrogen-bond, van der Waals, and stacking interactions.
By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH-π stacking. These issues are resolved by a combination of (15)N-(1)H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH-π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH-π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.
Adrenomedullin (AM) is a peptidic hormone that was isolated in 1993, the function of which is related to several diseases such as diabetes, hypertension, and cancer. Compound 1 is one of the first nonpeptidic small-molecule negative modulators of AM, identified in a high-throughput screen carried out at the National Cancer Institute. Herein we report the synthesis of a series of analogues of 1. The ability of the synthesized compounds to disrupt the binding between AM and its monoclonal antibody has been measured, together with surface plasmon resonance (SPR)-based binding assays as implemented with Biacore technology. These data were used to derive a three-dimensional quantitative structure-activity relationship (3D-QSAR) model, with a q(2) (LOO) value of 0.8240. This study has allowed us to identify relevant features for effective binding to AM: the presence of a hydrogen-bond donor group and an aromatic ring. Evaluation of the ability of selected compounds to modify cAMP production in Rat2 cells showed that the presence of a free carboxylic acid is essential for negative AM modulation.
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