Cell Permeability and Target Engagement of Middle-Sized Molecules Quantified by In-Cell NMR

Middle-sized molecules, such as macrocycles and cyclic peptides, are gaining attention as novel drug modalities due to their ability to permeate into cells and interfere with intracellular protein-protein interactions (PPIs). However, predicting and measuring the cell permeability of middle-sized molecules remains challenging, and there is only limited data about intracellular target-specific engagement. Here, we developed a floating in-cell NMR strategy to quantify cell permeability and intracellular target-specific engagement of middle-sized molecules in living mammalian cells. Compared to conventional in-cell NMR methods relying on gel encapsulation of cells, the floating strategy enhanced the sensitivity of the NMR measurement while maintaining cell survival and avoiding potential concerns of trapping middle-sized molecules in gel materials. Using this approach, we successfully measured the cell permeation rates and apparent intracellular target affinities of a hydrophobic middle-sized molecule, FK506. An increase in temperature substantially accelerated cell permeation without affecting intracellular affinity, suggesting temperature-dependent changes in membrane fluidity that specifically affect the cell permeability of FK506. The in-cell NMR approach developed here was also applicable to another middle-sized molecule, rapamycin. Our strategy provides quantitative pharmacodynamic data without any chemical modifications, offering valuable insights for developing middle-sized molecules that are effective for intracellular PPIs.