Background: rhodamines are dyes widely used as fluorescent tags in cell imaging, probing of mitochondrial membrane potential, and as P-glycoprotein model substrates. In all these applications, detailed understanding of the interaction between rhodamines and biomembranes is fundamental. Methods: we combined atomistic molecular dynamics (MD) simulations and fluorescence spectroscopy to characterize the interaction between rhodamines 123 and B (Rh123 and RhB, respectively) and POPC bilayers. Results: while the xanthene moiety orients roughly parallel to the membrane plane in unrestrained MD simulations, variations on the relative position of the benzoic ring (below the xanthene for Rh123, above it for RhB) were observed, and related to the structure of the two dyes and their interactions with water and lipids. Subtle distinctions were found among different ionization forms of the probes. Experimentally, RhB displayed a lipid/water partition coefficient more than two orders of magnitude higher than Rh123, in agreement with free energy profiles obtained from umbrella sampling MD. Conclusions: this work provided detailed insights on the similarities and differences in the behavior of bilayer-inserted Rh123 and RhB, related to the structure of the probes. The much higher affinity of RhB for the membranes increases the local concentration and explains its higher apparent affinity for P-glycoprotein reconstituted in model membranes.

The transport of drugs by efflux transporters in biomembranes limits their bioavailability and is a major determinant of drug resistance development by cancer cells and pathogens. A large number of chemically dissimilar drugs are transported, and despite extensive studies, the molecular determinants of substrate specificity are still not well understood. In this work, we explore the role of polar and non-polar interactions on the interaction of a homologous series of fluorescent amphiphiles with the efflux transporter P-glycoprotein. The interaction of the amphiphiles with P-glycoprotein is evaluated through effects on ATPase activity, efficiency in inhibition of [¹²⁵I]-IAAP binding, and partition to the whole native membranes containing the transporter. The results were complemented with partition to model membranes with a representative lipid composition, and details on the interactions established were obtained from MD simulations. We show that when the total concentration of amphiphile is considered, the binding parameters obtained are apparent and do not reflect the affinity for P–gp. A new formalism is proposed that includes sequestration of the amphiphiles in the lipid bilayer and the possible binding of several molecules in P–gp’s substrate-binding pocket. The intrinsic binding affinity thus obtained is essentially independent of amphiphile hydrophobicity, highlighting the importance of polar interactions. An increase in the lipophilicity and amphiphilicity led to a more efficient association with the lipid bilayer, which maintains the non-polar groups of the amphiphiles in the bilayer, while the polar groups interact with P–gp’s binding pocket. The presence of several amphiphiles in this orientation is proposed as a mechanism for inhibition of P-pg function.

The association of small molecules with lipid membranes plays a central role in drug delivery, pharmacokinetics, toxicity, and membrane biophysics, also being of fundamental importance in drug pharmacodynamics given that most drug targets are membrane-associated proteins. Accurate determination of solute–membrane association affinities, however, remains challenging due to the diversity of experimental systems, the complexity of membrane environments, and the intrinsic limitations of individual methodologies. This review provides a comprehensive overview of the experimental and computational approaches currently used to quantify small molecules association with lipid membranes. Standard experimental techniques, including spectroscopy-based methods, calorimetry, electrophoretic measurements, and surface-sensitive approaches, are discussed alongside established computational strategies ranging from continuum models to atomistic molecular dynamics simulations. Particular emphasis is placed on the formalisms required for data analysis, including partitioning models and thermodynamic frameworks, as well as on the assumptions underlying each method. The validity limits, sources of uncertainty, and common experimental and interpretative pitfalls are critically examined. By providing a unified and comparative perspective, this work establishes a structured framework for the quantitative study of solute–membrane interactions, guiding new researchers in the selection of appropriate methodologies and in the rigorous analysis of experimental and computational results. Moreover, it enables the consistent and quantitative rationalization of affinity parameters reported across the literature, supporting the development of curated datasets and predictive relationships that can inform the design of new and more effective drugs.

The association of small molecules with lipid membranes plays a central role in drug delivery, pharmacokinetics, toxicity, and membrane biophysics, also being of fundamental importance in drug pharmacodynamics given that most drug targets are membrane-associated proteins. Accurate determination of solute–membrane association affinities, however, remains challenging due to the diversity of experimental systems, the complexity of membrane environments, and the intrinsic limitations of individual methodologies. This review provides a comprehensive overview of the experimental and computational approaches currently used to quantify small molecule association with lipid membranes. Standard experimental techniques, including spectroscopy-based methods, calorimetry, electrophoretic measurements, and surface-sensitive approaches, are discussed alongside established computational strategies ranging from continuum models to atomistic molecular dynamics simulations. Particular emphasis is placed on the formalisms required for data analysis, including partitioning models and thermodynamic frameworks, as well as on the assumptions underlying each method. The validity limits, sources of uncertainty, and common experimental and interpretative pitfalls are critically examined. By providing a unified and comparative perspective, this work establishes a structured framework for the quantitative study of solute–membrane interactions, guiding new researchers in the selection of appropriate methodologies and in the rigorous analysis of experimental and computational results. Moreover, it enables the consistent and quantitative rationalization of affinity parameters reported across the literature, supporting the development of curated datasets and predictive relationships that can inform the design of new and more effective drugs.