Spying on FABPs: characterising protein–ligand interactions with 19F NMR assays

Fatty acid binding proteins play a pivotal role in lipid transport and signalling, with fatty acid binding protein 4 (FABP4) emerging as a promising target for metabolic and inflammatory diseases. This work describes the development and validation of a 19F Carr–Purcell–Meiboom–Gill (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 isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and 15N–1H heteronuclear single quantum correlation (HSQC), with good correlation across methods.