2022-09-232022-09-232022Monaco, S., Ramírez Cárdenas, J., Carmona Asenjo, A.T., Robina Ramírez, I. y Angulo Álvarez, J. (2022). Inter-Ligand STD NMR: An Efficient 1D NMR Approach to Probe Relative Orientation of Ligands in a Multi-Subsite Protein Binding Pocket. Pharmaceuticals, 15 (8), 1030.1424-8247https://hdl.handle.net/11441/137357In 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.application/pdf14 p.engAttribution-NonCommercial-NoDerivatives 4.0 Internacionalhttp://creativecommons.org/licenses/by-nc-nd/4.0/Fragment Based Drug DiscoveryLigand-based NMRMulti-frequency STD NMRMulti-subsite binding pocketsProtein-ligand interactionsSaturation transfer difference NMRinfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccesshttps://doi.org/10.3390/ph15081030