Figure 1. Schematic representation of the most common NMR‐based approaches in FBDD. (a) Chemical shift mapping (bottom panel) and SAR by the NMR approach (top panel). A first ligand binding in a first site can be discovered by screening compounds in mixtures from a library of scaffolds and monitoring chemical shift changes typically in two‐dimensional – or – spectra of uniformly or selectively labeled protein targets. Following the identification of the first fragment (F1), a second screen is carried out in the presence of this fragment against a second scaffold library. By using chemical shift mapping, compounds can be identified that can simultaneously bind an adjacent region within the F1 binding site. The bottom panels illustrate this process: first, the spectrum of the target is collected in its apo form (left panel, blue cross‐peaks) and compared to (overlaid on) the same spectrum recorded in the presence of test ligands (left panel, purple cross‐peaks). The nature of the cross‐peak movements enables first the identification and then the rough location of the binding site for a first ligand F1. Subsequently, the spectrum of the target in complex with F1 is collected (bottom right, purple cross‐peaks) and compared to (overlaid on) the same spectrum recorded in the presence of F2 test ligands (bottom right, red cross‐peaks). Following structural characterization by NMR of the ternary complex target–F1–F2, high‐affinity bidentate compounds can be designed by covalently linking the two fragments. (b) Schematic illustration of the SAR by ILOE approach in which protein‐mediated ILOEs between two binding fragments (F1 and F2) on the surface of a given unlabeled drug target are used to screen for a pair of binders. Proton–proton magnetization transfers can be detected with a two‐dimensional – NOESY of the two test ligands (or mixtures of test fragments at concentrations varying from 100 µM to 1 mM) measured in the presence of a substoichiometric amount of target (typically at a concentration of 1–10 µM). With this method, low‐affinity ligands (micro‐ to millimolar) can be identified by the presence of ILOE cross‐peaks (bottom panel) if their internuclear distance is less than 5Å . (c) Schematic representation of the paramagnetic enhancement approach for the detection of second‐site binders by using a spin‐labeled first ligand (F2*). In the one‐dimensional T _1ρ experiment (bottom panel), the binding of a test ligand is manifested by a decrease of signal intensity in one‐dimensional ¹H NMR spectra of the test compound (or a mixture of test compounds; usually at concentrations from 100 µM to 1 mM) in the presence of a substoichiometric amount of protein target (typically at a concentration of 1–10 µM). In performing a second‐site screen, this decrease in signal intensity is enhanced if the F1 ligand is close in space (less than 10 Å) to the spin‐labeled F2* compound (present at protein‐saturating concentrations; bottom panel).