In recent years, Saturation Transfer Difference NMR (STD NMR) has been proven to be a powerful and versatile ligand-based NMR technique to elucidate crucial aspects in the investigation of protein-ligand complexes. Novel STD NMR approaches relying on “multi-frequency” irradiation have enabled us to even elucidate specific ligand-amino acid interactions and explore the binding of fragments in previously unknown binding subsites. Exploring multi-subsite protein binding pockets is especially important in Fragment Based Drug Discovery (FBDD) to design leads of increased specificity and efficacy. We hereby propose a novel multi-frequency STD NMR approach based on direct irradiation of one of the ligands in a multi-ligand binding process, to probe the vicinity and explore the relative orientation of fragments in adjacent binding sub-sites, which we called Inter-Ligand STD NMR (IL-STD NMR). We proved its applicability on (i) a standard protein-ligand system commonly used for ligand-observed NMR benchmarking: Naproxen as bound to Bovine Serum Albumin, and (ii) the biologically relevant system of Cholera Toxin Subunit B and two inhibitors adjacently bound within the GM1 binding site. Relative to Inter-Ligand NOE (ILOE), the current state-of-the-art methodology to probe relative orientations of adjacent ligands, IL-STD NMR requires about one tenth of the experimental time and protein consumption, making it a competitive methodology with the potential to be applied in the pharmaceutical industries.

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. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

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 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. However, the use of the STD NMR technique together with full relaxation matrix calculations for the validation of 3D structures of protein-ligand complexes remains a major milestone in the field. In this work, we present a new approach based on a reduced relaxation matrix that allows very fast 3D structure evaluation of lowaffinity protein-ligand complexes by STD NMR data and molecular dynamics simulations.

STD NMR is a powerful ligand-based tool for screening small molecules and low molecular weight fragments for their interaction with a given macromolecule, and it has become the spectroscopic technique of choice for the study of medium/weak affinity protein-ligand interactions. In the pharmaceutical industry, there is a great interest in the accurate and fast determination of protein-fragment binding affinities, typically low. STD NMR is a uniquely suited technique to accurately determine weak proteinligand affinities. However, a drawback of the technique is that, in order to gain quantitative structural or affinity information from STD NMR experiments, long series of experiments at increasing values of the saturation time of the protein must be carried out, to get the full analysis of the so-called STD NMR build-up curve (“initial slopes approach”). To resolve this limitation, we have developed a protocol that allows to get accurate initial slopes using STD NMR data acquired at only 2 saturation times. We demonstrate that our protocol, called the Reduced Dataset STD NMR approach (rd-STD NMR), allows the very fast determination of dissociation constants of low affinity protein-ligand interactions.