Passive Transport across Cell Membranes beyond the Overton Rule: Insights from Solute Exchange in Vesicles and Molecular Dynamics of Atropisomers

Bioavailability of a drug is critically dependent on its cell membrane permeability. Empirical rules guiding drug design consolidated the dogma that large molecules cannot cross cell membranes by passive diffusion. However, the more amphiphilic atropisomers of redaporfin, an 1135 Da bacteriochlorin photosensitizer used in photodynamic therapy, exhibited fast cell uptake and high photodynamic activity in vitro. This motivated detailed studies of redaporfin atropisomers and their interactions with cell membrane models. Experimental studies on membrane affinity, permeation rates, and exchange dynamics were complemented by molecular dynamics simulations, to reveal the nature of the interactions between the atropisomers and lipid bilayers, the orientation and location of the membrane-bound atropisomers, free energy profiles, and mechanisms governing membrane permeation. Our results indicate that the asymmetric distribution of the meso-phenyl sulfonamide groups (atropisomer α4) generates a large amphiphilic moment. This enhances its membrane affinity and positions the bacteriochlorin ring deeper in the membrane. However, these strong membrane interactions result in a slow exchange of α4 between lipid membranes, restricting its distribution in complex, membrane-rich environments. In contrast, the more symmetrical atropisomer αβαβ exhibits approximately 10-fold lower membrane affinity and localizes closer to the membrane-water interface. This weaker interaction facilitates rapid exchange between membranes, occurring within minutes at 37 °C. Molecular dynamics simulations reveal relatively low energy barriers for membrane translocation, consistent with experimentally estimated fast translocation. Distinct permeation mechanisms were observed for the two atropisomers, providing insights into their differential behavior in passive membrane transport. In particular, the fast cell uptake of the α4 atropisomer is properly described by the bind-flip mechanism, where the sulfonamide groups first approach the bilayer in a "binding" mode, and then the molecule "flips" to place the macrocycle in a more internal position. Our results show how amphiphilicity and conformation flexibility are critical determinants in the cellular internalization of large molecules.