Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photocatalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.

Carbohydrate-protein interactions are key for cell-cell and host-pathogen recognition and thus, emerged as viable therapeutic targets. However, their hydrophilic nature poses major limitations to the conventional development of drug-like inhibitors. To address this shortcoming, four fragment libraries were screened to identify metal-binding pharmacophores (MBPs) as novel scaffolds for inhibition of Ca2+-dependent carbohydrate-protein interactions. Here, we show the effect of MBPs on the clinically relevant lectins DC-SIGN, Langerin, LecA and LecB. Detailed structural and biochemical investigations revealed the specificity of MBPs for different Ca2+-dependent lectins. Exploring the structure-activity relationships of several fragments uncovered the functional groups in the MBPs suitable for modification to further improve lectin binding and selectivity. Selected inhibitors bound efficiently to DC-SIGN-expressing cells. Altogether, the discovery of MBPs as a promising class of Ca2+-dependent lectin inhibitors creates a foundation for fragment-based ligand design for future drug discovery campaigns.

DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) is a pattern recognition receptor expressed on immune cells and involved in the recognition of carbohydrate signatures present on various pathogens, including HIV, Ebola, and SARS-CoV-2. Therefore, developing inhibitors blocking the carbohydrate-binding site of DC-SIGN could generate a valuable tool to investigate the role of this receptor in several infectious diseases. Herein, we performed a fragment-based ligand design using 4-quinolone as a scaffold. We synthesized a library of 61 compounds, performed a screening against DC-SIGN using an STD reporter assay, and validated these data using protein-based 1 H− 15 N HSQC NMR. Based on the structure−activity relationship data, we demonstrate that ethoxycarbonyl or dimethylaminocarbonyl in position 2 or 3 is favorable for the DC-SIGN binding activity, especially in combination with fluorine, ethoxycarbonyl, or dimethylaminocarbonyl in position 7 or 8. Furthermore, we demonstrate that these quinolones can allosterically modulate the carbohydrate binding site, which offers an alternative approach toward this challenging protein target.

Langerin is a mammalian C-type lectin expressed on Langerhans cells in the skin. As an innate immune cell receptor, Langerin is involved in coordinating innate and adaptive immune responses against various incoming threats. We have previously reported a series of thiazolopyrimidines as murine Langerin ligands. Prompted by the observation that its human homologue exhibits different binding specificities for these small molecules, we report here our investigations to define their exact binding site. By using structural comparison and molecular dynamics simulations, we showed that the nonconserved short loops have a high degree of conformational flexibility between the human and murine homologues. Sequence analysis and mutational studies indicated that a pair of residues are essential for the recognition of the thiazolopyrimidines. Taking solvent paramagnetic relaxation enhancement NMR studies together with a series of peptides occupying the same site, we could define the cleft between the short and long loops as the allosteric binding site for these aromatic heterocycles.

Two orthogonal, metal free click reactions, enabled to glycosylate ubiquitin and its mutant A28C forming two protein scaffolds with high affinity for BambL, a lectin from the human pathogen Burkholderia...

The Cover Feature illustrates the photo‐initialization by UV light of the thiol‐ene reaction between the alkene of a newly designed paramagnetic tag and the thiol group of the cysteine residue on a model protein. The protein tagging occurs in 1 hour. We tagged the protein with three different lanthanide ions, which induced different chemical shifts on the protein NMR signals. Paramagnetic restraints were measured and the tensor calculated. More information can be found in the Article by L. Cerofolini, C. Nativi and co‐workers.

The increasing diffusion of antimicrobial resistance (AMR) across more and more bacterial species emphasizes the urgency of identifying innovative treatment strategies to counter its diffusion. Pathogen infection prevention is among the most effective strategies to prevent the spread of both disease and AMR. Since their discovery, vaccines have been the strongest prophylactic weapon against infectious diseases, with a multitude of different antigen types and formulative strategies developed over more than a century to protect populations from different pathogens. In this review, we review the main characteristics of vaccine formulations in use and under development against AMR pathogens, focusing on the importance of administering multiple antigens where possible, and the challenges associated with their development and production. The most relevant antigen classes and adjuvant systems are described, highlighting their mechanisms of action and presenting examples of their use in clinical trials against AMR. We also present an overview of the analytical and formulative strategies for multivalent vaccines, in which we discuss the complexities associated with mixing multiple components in a single formulation. This review emphasizes the importance of combining existing knowledge with advanced technologies within a Quality by Design development framework to efficiently develop vaccines against AMR pathogens.

Ubiquitin as <i>scaffold</i> <i>protein</i> and aryl-α-O-fucoside as determinant to achieve conceptually new ligands with high affinity for <i>Burkholderia ambifaria </i>lectin are described.<div>Ub mutant A28C which displays a Cys residue in addition to the eight Lys residues was expressed. </div><div>The resulting <i>scaffold</i> was orthogonally glycosylated </div><div>to display one thio-rhamnose and up to eight residues of aryl-α-O-fucoside. </div><div>The glycosylated Ub-based scaffolds have unprecedented immune properties.<br></div>