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.