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. Comparison of relaxation and exchange matrix calculations with ¹H STD NMR experimental data is essential for the validation of 3D structures of protein-ligand complexes. In this work, we present a new approach based on the calculation of a reduced relaxation matrix, in combination with funnel metadynamics MD simulations, that allows a very fast generation of experimentally STD-NMR-validated 3D structures of low-affinity protein-ligand complexes.

STD NMR spectroscopy is a powerful ligand-observed NMR tool for screening and characterizing the interactions of small molecules and low molecular weight fragments with a given macromolecule, identifying the main intermolecular contacts in the bound state. It is also a powerful analytical technique for the accurate determination of protein-ligand dissociation constants (K_D) of medium-to-weak affinity, of interest in the pharmaceutical industry. However, accurate K_D determination and epitope mapping requires a long series of experiments at increasing saturation times to carry out a full analysis using the so-called STD NMR build-up curve approach and apply the "initial slopes approximation". Here, we have developed a new protocol to bypass this important limitation, which allows us to obtain initial slopes by using just two saturation times and, hence, to very quickly determine precise protein-ligand dissociation constants by STD NMR.

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.

Bromodomain-containing protein 9 (BRD9) belongs to the non-canonical BAF chromatin remodeling complex and represents a relevant therapeutic target in pathologies featuring dysregulated epigenetic control. The absence of clinically validated inhibitors and the need for diversified chemical entities highlight the interest in identifying new scaffolds targeting this protein. In this study, Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) was employed to assess its suitability for characterizing BRD9–ligand interactions within a fragment-based discovery framework. STD NMR conditions were first optimized using the known BRD9 ligand 1, verifying the presence of interaction signals. A pharmacophore-based virtual screening campaign was then performed using libraries of commercially available fragments, leading to the selection of a novel isatin derivative, i.e., compound 2, whose binding was demonstrated in AlphaScreen assays. STD NMR experiments provided epitope mapping consistent with the predicted binding mode, thus supporting the stability of the interaction in solution. Moreover, a competitive STD experiment demonstrated displacement of 2 by a reference ligand, confirming the binding within the canonical BRD9 pocket. Overall, this study establishes STD NMR as a reliable approach for probing BRD9–ligand interactions and for the identification and validation of BRD9-targeting scaffolds suitable for future structure-guided optimization.

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.

Saturation transfer difference nuclear magnetic resonance (STD NMR) spectroscopy is a powerful and robust technique for the characterization of receptor–ligand interactions of medium to weak affinity in solution, providing binding epitope maps that reveal structural details of these interactions at atomic level. This knowledge is essential for understanding the structural basis of biomolecular recognition processes of biological relevance and plays a key role in drug discovery. Temperature is a critical factor influencing the sensitivity of STD NMR spectroscopy, as it directly affects the kinetics of complex formation, thereby modulating the observed STD effects. In this study, we systematically investigated the impact of temperature on STD NMR‐derived binding epitope maps. Our findings highlight that, whenever protein stability permits, temperature should be considered an essential experimental parameter to be optimized for each system to obtain binding epitope maps with well‐discriminated protein–ligand contact information. For the example cases studied herein, we demonstrate that temperature optimization allows to better assess the contribution of each individual contact to protein–ligand binding (enhanced ligand epitope mapping discrimination).