Parallelized 61-Sample Inhibition Screening Accelerated by ¹⁹F-MRI Compressed Sensing

¹⁹F NMR is a well-established technique for screening ligands in early drug discovery. However, conventional NMR measurements are inherently sequential, which limits experimental throughput. We recently demonstrated that ¹⁹F MR imaging (MRI) enables spatial parallelization of such measurements. In this study, we leverage the reporter assay format, where the transverse relaxation ( $T_2$ ) of a fluorinated reporter ligand is monitored in the presence of potential competitors. Using $T_2$ -weighted MR images, up to 61 miniaturized samples can be resolved simultaneously. The screening assay operates at microliter-scale volumes in a custom miniaturized honeycomb sample holder, providing a readily accessible and scalable quantitative binding assay. To further accelerate data acquisition, we developed optimized imaging protocols based on compressed sensing (CS), enabling substantial reductions in measurement time without compromising quantitative accuracy. This approach allows parallel screening of 55 potential ligands and efficient determination of relative binding affinities through simultaneous dose-response experiments. We demonstrate the method on the model protein trypsin and the antibiotic target PPAT, characterizing several ligands with distinct binding properties. This study highlights the potential of MRI to enhance throughput and efficiency in early-stage drug discovery markedly.