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

Evgenia Konstantinidou; Indu Chandrashekaran; Menachem J Gunzburg; Naureen Akhtar; Bradley Doak; Martin Scanlon

Just Accepted

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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

Evgenia Konstantinidou, Indu Chandrashekaran, Menachem J Gunzburg, Naureen Akhtar, Bradley Doak, Martin Scanlon

Abstract

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.

CH25102 Accepted 01 October 2025