Quantitative Competition Binding of Fluorinated Ligands by Real‐Time 19 F In‐Cell NMR to Assess Isoform Selectivity in Human Cells

Drug development is a risky endeavour with a high failure rate, often caused by the limited ability to predict the efficacy and interactions of candidate drugs in a native cellular environment. In this context, in-cell NMR spectroscopy is a promising tool for assessing drug-target binding directly in living cells, thereby improving the screening and development of new molecules. In this study, we used real-time in-cell 19F NMR spectroscopy in a flow bioreactor to observe competitive binding of fluorinated benzenesulfonamide derivatives to three cytosolic isoforms of carbonic anhydrase. Quantitative measurement of the dissociation constants relative to a spy ligand allowed an accurate ranking of the compounds based on their intracellular affinities for each isoform. The use of two fluorinated ligands allowed simultaneous observation of spy ligand displacement and test ligand binding, as well as estimation of the effective ratio of free ligand concentrations under poor solubility conditions. We also show that signal saturation caused by short repetition times, which can significantly impact the analysis, can be easily corrected a posteriori. Overall, we show that real-time in-cell 19F NMR spectroscopy can reliably quantify drug-target binding in the cellular environment, paving the way for future applications in drug discovery.