On-Cell Saturation Transfer Difference NMR Spectroscopy on Ion Channels: Characterizing Negative Allosteric Modulator Binding Interactions of P2X7.

P2X7 receptors are important drug targets involved in pathologies ranging from psychiatric disorders to cancer. Being membrane embedded receptors, they are more challenging for structural characterization, and at present, we only have a small number of X-ray and cryo-EM structures for P2X7 bound to antagonists. We demonstrate that saturation transfer difference (STD) NMR on live mammalian cells (on-cell STD NMR) overexpressing P2X7 receptors allows further structural insight on the complexes of P2X7 with two potent negative allosteric modulators, namely, AZ10606120 and JNJ-47965567, via the determination of the binding epitope mapping of the interactions, e.

The chemical instability of the survivin inhibitor - sepantronium bromide (YM155) studied by stimulated Raman, NMR and UV-Vis spectroscopies.

Sepantronium bromide, which shows a broad spectrum of anticancer action, is allegedly chemically unstable. This instability might significantly limit the final antineoplastic efficacy of the drug. Here, we report our studies on these chemical stability issues under different chemical environments using advanced spectroscopies.

STD NMR Epitope Perturbation by Mutation Unveils the Mechanism of YM155 as an Arginine-Glycosyltransferases Inhibitor Effective in Treating Enteropathogenic Diseases.

Enteropathogenic arginine-glycosyltransferases (Arg-GTs) alter higher eukaryotic proteins by attaching a GlcNAc residue to arginine acceptor sites, disrupting essential pathways such as NF-κB signaling, which promotes bacterial survival. These enzymes are potential drug targets for treating related diseases. In this study, we present a novel STD NMR Epitope Perturbation by Mutation spectroscopic approach that, in combination with hydrogen-deuterium exchange mass spectrometry (HDX-MS), and molecular dynamics simulations, shows that the highly potent broad-spectrum anticancer drug YM155 serves as a potential noncompetitive inhibitor of these enzymes.

3,3'-Diindolylmethane (DIM): A Molecular Scaffold for Inhibition of WWP1 and WWP2, Members of the NEDD4 Family HECT E3 Ligases.

Indole-3-carbinol (I3C) is a metabolic derivative of glucobrassicin found in cruciferous vegetables. Known for its anticarcinogenic properties, I3C has been shown to target the NEDD4 family HECT E3 ligases, NEDD4-1 and WWP1, yet confirmation for the latter is lacking. Here, we characterize the interactions of I3C and a set of 17 derivatives with WWP1 and its homologue, WWP2.

α-GalCer sp-iminoglycolipid analogs as CD1d-dependent iNKT modulators: Evaluation of their immunotherapeutic potential in murine models of asthma and autoimmune hepatitis.

Invariant natural killer T (iNKT) cells are a subset of innate T cells displaying powerful immunomodulatory functions. Despite extensive preclinical research on the use of iNKT agonist and antagonist for various diseases, translating these findings into successful clinical applications has proven challenging, leaving no approved treatments to date. Efforts to optimize therapeutic outcomes by developing alternative glycolipids to α-galactosylceramide (α-GalCer or KRN7000), the prototypical iNKT antigen, have shown improved preclinical results.

Multifrequency-STD NMR unveils the first Michaelis complex of an intramolecular trans-sialidase from Ruminococcus gnavus.

RgNanH is an intramolecular trans-sialidase expressed by the human gut symbiont Ruminococcus gnavus, to utilise intestinal sialylated mucin glycan epitopes. Its catalytic domain, belonging to glycoside hydrolase GH33 family, cleaves off terminal sialic acid residues from mucins, releasing 2,7-anhydro-Neu5Ac which is then used as metabolic substrate by R. gnavus to proliferate in the mucosal environment.

Fluorinated trehalose analogues for cell surface engineering and imaging of .

The sensitive, rapid and accurate diagnosis of () infection is a central challenge in controlling the global tuberculosis (TB) pandemic. Yet the detection of mycobacteria is often made difficult by the low sensitivity of current diagnostic tools, with over 3.6 million TB cases missed each year.

Fast Quantitative Validation of 3D Models of Low-Affinity Protein-Ligand Complexes by STD NMR Spectroscopy.

Low-affinity protein-ligand interactions are important for many biological processes, including cell communication, signal transduction, and immune responses. Structural characterization of these complexes is also critical for the development of new drugs through fragment-based drug discovery (FBDD), but it is challenging due to the low affinity of fragments for the binding site. Saturation transfer difference (STD) NMR spectroscopy has revolutionized the study of low-affinity receptor-ligand interactions enabling binding detection and structural characterization.

Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes.

Fluorinated Man as a High Mannose Mimetic to Unravel Its Recognition by DC-SIGN Using NMR.

Lectins are capable of reading out the structural information contained in carbohydrates through specific recognition processes. Determining the binding epitope of the sugar is fundamental to understanding this recognition event. Nuclear magnetic resonance (NMR) is a powerful tool to obtain this structural information in solution; however, when the sugar involved is a complex oligosaccharide, such as high mannose, the signal overlap found in the NMR spectra precludes an accurate analysis of the interaction.

Glycosaminoglycans: What Remains To Be Deciphered?

Glycosaminoglycans (GAGs) are complex polysaccharides exhibiting a vast structural diversity and fulfilling various functions mediated by thousands of interactions in the extracellular matrix, at the cell surface, and within the cells where they have been detected in the nucleus. It is known that the chemical groups attached to GAGs and GAG conformations comprise "glycocodes" that are not yet fully deciphered. The molecular context also matters for GAG structures and functions, and the influence of the structure and functions of the proteoglycan core proteins on sulfated GAGs and vice versa warrants further investigation.

FUT8-Directed Core Fucosylation of N-glycans Is Regulated by the Glycan Structure and Protein Environment.

FUT8 is an essential α-1,6-fucosyltransferase that fucosylates the innermost GlcNAc of N-glycans, a process called core fucosylation. , FUT8 exhibits substrate preference for the biantennary complex N-glycan oligosaccharide (G0), but the role of the underlying protein/peptide to which N-glycans are attached remains unclear. Here, we explored the FUT8 enzyme with a series of N-glycan oligosaccharides, N-glycopeptides, and an Asn-linked oligosaccharide.

NleB/SseK-catalyzed arginine-glycosylation and enteropathogen virulence are finely tuned by a single variable position contiguous to the catalytic machinery.

NleB/SseK effectors are arginine-GlcNAc-transferases expressed by enteric bacterial pathogens that modify host cell proteins to disrupt signaling pathways. While the conserved NleB and NleB1 proteins display a broad selectivity towards host proteins, SseK1, SseK2, and SseK3 have a narrowed protein substrate selectivity. Here, by combining computational and biophysical experiments, we demonstrate that the broad protein substrate selectivity of NleB relies on Tyr284, a second-shell residue contiguous to the catalytic machinery.