Monaco Serena, Ramírez-Cárdenas Jonathan, Carmona Ana Teresa, Robina Inmaculada, Angulo Jesus
School of Pharmacy, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
Instituto de Investigaciones Químicas (CSIC-Universidad de Sevilla), 41092 Seville, Spain.
Pharmaceuticals (Basel). 2022 Aug 21;15(8):1030. doi: 10.3390/ph15081030.
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 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.
近年来，饱和转移差分核磁共振（STD NMR）已被证明是一种强大且通用的基于配体的核磁共振技术，可用于阐明蛋白质 - 配体复合物研究中的关键方面。依赖于“多频”照射的新型STD NMR方法使我们能够阐明特定的配体 - 氨基酸相互作用，并探索片段在以前未知的结合亚位点中的结合情况。在基于片段的药物发现（FBDD）中，探索多亚位点蛋白质结合口袋对于设计具有更高特异性和功效的先导化合物尤为重要。我们在此提出一种基于在多配体结合过程中直接照射其中一种配体的新型多频STD NMR方法，以探测相邻结合亚位点中片段的附近区域并探索其相对取向，我们将其称为STD NMR（IL - STD NMR）。我们证明了它在以下方面的适用性：（i）一种常用于配体观测核磁共振基准测试的标准蛋白质 - 配体系统：萘普生与牛血清白蛋白结合，以及（ii）霍乱毒素亚基B与GM1结合位点内相邻结合的两种抑制剂的生物学相关系统。相对于用于探测相邻配体相对取向的当前最先进方法——配体间核Overhauser效应（ILOE），IL - STD NMR所需的实验时间和蛋白质消耗量约为其十分之一，使其成为一种有竞争力的方法，具有应用于制药行业的潜力。
