Quantification and characterization of P-glycoprotein-substrate interactions

It is generally accepted that P-glycoprotein binds its substrates in the lipid phase of the membrane. Quantification and characterization of the lipid-transporter binding step are, however, still a matter of debate. We therefore selected 15 structurally diverse drugs and measured the binding constants from water to the activating (inhibitory) binding region of P-glycoprotein, K-tw(1) (K-tw(2)), as well as the lipid-water partition coefficients, K-lw. The former were obtained by measuring the concentrations of half-maximum activation (inhibition), K-1 (K-2), in living NIH-MDR-G 185 mouse embryo fibroblasts using a Cytosensor microphysiometer, and the latter were derived from surface activity measurements. This allowed determination of the membrane concentration of drugs at half-maximum P-glycoprotein activation (C-b(1) = (0.02 to 67) mmol/L lipid), which is much higher than the corresponding aqueous concentration (K-1 = (0.02 to 376) mu M). Moreover we determined the free energy of drug binding from water to the activating binding region of the transporter (Delta G degrees(tw(1)) = (-30 to -54) kJ/mol), the free energy of drug partitioning into the lipid membrane (Delta G degrees(lw) = (-23 to -34) kJ/mol), and, as the difference of the two, the free energy of drug binding from the lipid membrane to the activating binding region of the transporter (Delta G degrees(tl(1)) = (-7 to -27) kJ/mol). For the compounds tested AG degrees(tl(1)) was less negative than Delta G degrees(tl(1)) but varied more strongly. The free energies of substrate binding to the transporter within the lipid phase, Delta G degrees(tl(1)), are consistent with a modular binding concept, where the energetically most efficient binding module comprises two hydrogen bond acceptor groups.