High-Resolution Relaxometry for Fragment Screening

Abstract

Characterizing weak molecular interactions remains a major challenge in drug discovery. Here, we demonstrate the power of high-resolution relaxometry (HRR), using a fast sample shuttle system, as a highly sensitive ligand-based screening method. By measuring longitudinal relaxation rates over a wide magnetic field range (2–600 MHz), we obtained site-specific Nuclear Magnetic Resonance Dispersion (NMRD) profiles for three ligands interacting with the catalytic domain of Human Matrix Metalloproteinase-12 (MMP-12). HRR unambiguously detects weak binding (K D ≈ 10–6–10–4) at protein concentrations as low as 2 μM. The analysis of the NMRD profiles provides direct, quantitative assessment of the complex dynamics, yielding a rotational correlation time in excellent agreement with the protein hydrodynamic properties. This enables discrimination between genuine protein–ligand binding and alternative processes like ligand aggregation, often observed in pan-assay interference compounds (PAINS). HRR therefore provides a robust, low-sample-consumption, physicochemical approach for fragment screening and characterization of protein–ligand complex dynamics.