Interaction of a Homologous Series of Amphiphiles with P-glycoprotein in a Membrane Environment—Contributions of Polar and Non-Polar Interactions

Abstract

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.

Ligand’s Partition to the Lipid Bilayer Should Be Accounted for When Estimating Their Affinity to Proteins

“…The former analysis is preferable when the molar concentration of the protein is not well known (only its total mass is known, for example) or when the ligand occupancy number in the protein is not well defined (as is the case for proteins with large binding pockets corresponding to several possibly overlapping binding sites). The comparison between the two formalisms may provide information on the number of ligands that can bind to the protein binding pocket, as was recently carried out for the interaction of a series of ligands to P-glycoprotein [ 15 ].…”

“…P-glycoprotein (P-gp) is an example of an intrinsic membrane protein, and the protein binding pocket is usually considered accessible by the ligand in the lipid portion of the membrane [ 12 , 13 , 14 ]. Therefore, for this protein, an increase in the ligand lipophilicity leads to an increase in the local concentration in the membrane, and an increase in the apparent affinity is usually observed [ 15 , 16 ]. On the other hand, for very lipophilic ligands, increasing the volume of the lipid phase may lead to a dilution of the ligand in the membrane, thus resulting in a decrease in the apparent affinity [ 17 ].…”

“…Focus was given to the importance of using the local concentration of ligand in each medium, instead of the usually considered overall ligand concentration. This formalism has been applied to the characterization of the interaction of a homologous series of ligands with P-glycoprotein [ 15 ] and allowed the characterization of the ligands’ intrinsic affinities. Importantly, the same intrinsic affinities were obtained when the interaction was characterized by two distinct assays (ATPase activity and ligand displacement), performed at different membrane concentrations (0.1 and 1 mg protein/mL, respectively).…”

“…For an accurate and quantitative analysis of ligand-protein interactions in the presence of lipid membranes, the partition of the ligand to the membrane must be known and included in the analysis [ 15 , 36 ]. It will be shown in this article that the quantitative evaluation of the ligand interaction with the lipid portion of the membrane is necessary for the quantitative evaluation of the affinity of the ligand to the protein.…”

“…It will be shown in this article that the quantitative evaluation of the ligand interaction with the lipid portion of the membrane is necessary for the quantitative evaluation of the affinity of the ligand to the protein. The characterization of the interaction of the ligand with the specific membrane is therefore of major importance and should not be simply estimated from its partition between water and octanol (see, e.g., [ 15 , 37 , 38 , 39 , 40 , 41 , 42 ] for discussions on the limitations of this approach).…”