Kinetic NMR screening: rapidly quantifying fast ligand dissociation in fragment mixtures using ¹⁹F relaxation dispersion

Ligand-observed NMR is a key tool for detecting weak protein-ligand interactions, but translating binding signals into meaningful affinity measurements remains difficult, limiting the ability to rank and prioritize compounds in drug development campaigns. Here, we propose a conceptual shift by using ligand-observed 19F NMR R1ρ relaxation dispersion to screen compounds by dissociation kinetics rather than binding thermodynamics. The first-order nature of ligand dissociation enables direct measurement in complex mixtures, and we have successfully extracted rate constants (3,500–65,000 s-1) from mixtures of up to 30 compounds across three protein targets. This kinetic approach delivers medicinal chemists a simple, actionable parameter, typically in under 30 minutes per compound. With open-source and user-friendly analysis software developed, we anticipate this approach will accelerate drug development campaigns by facilitating the screening, characterisation, and ranking of compound libraries.