Spying on FABPs: Characterising protein-ligand interactions with 19 F NMR assays Keywords: 19 F CPMG, NMR, FABP4, niflumic acid, spy molecule, competition assay, fragment screening, binding affinity measurement

Fatty acid binding proteins play a pivotal role in lipid transport and signalling, with FABP4 emerging as a promising target for metabolic and inflammatory diseases. This work describes the development and validation of a ¹⁹F CPMG NMR displacement assay tailored for fragment screening and lead optimisation against FABP4. The method employs fluorinated spy molecules to quantify ligand binding through changes in transverse relaxation, offering a solution-phase, label-free alternative to conventional techniques. A focused screen validated the assay's broad affinity window and alignment with orthogonal data. The assay was benchmarked against ITC, SPR, and ¹⁵N-¹H HSQC, with good correlation across methods.