Inhibitors of multidrug efflux transporters : their membrane and protein interactions

Modulators and inhibitors of multidrug efflux transporters, like P-glycoprotein, are used to reduce or inhibit multidrug resistance, MDR, which leads to a failure of the chemotherapy of e.g. cancers, epilepsy, bacterial, parasitic, and fungal diseases. Binding and transport of first-, second-, and third-generation modulators and inhibitors of P-glycoprotein are discussed, taking into account the properties of the drug (H-bonding potential, dimensions, and pK(a) values) as well as the properties of the membrane

The rate of P-glycoprotein activation depends on the metabolic state of the cell

P-glycoprotein ATPase activity has been studied almost exclusively by measuring inorganic phosphate release from inside-out cellular vesicles. We have recently proposed a new method based on measurements of the extracellular acidification rate (ECAR) of living cells with a Cytosensor microphysiometer. This method allows for systematic investigation of the various factors influencing P-glycoprotein activation in living cells. Basal metabolic rates or ECARs of different MDR1-transfected cell lines were compared with those of the Mdr1a(-/-)1b(-/-) knockout, MRP1-transfected, and corresponding wild-type cell lines. Basal ECARs of all cells were on the order of 10(7) protons/cell/s, whereby those of genetically modified cells were on average (over all cell lines) slightly lower than those of wild-type cells. The expression level of P-glycoprotein in MDR1-transfected cells had no influence on basal ECARs. Verapamil-induced ECARs were specific for MDR1-transfected cells and increased with the expression level of P-glycoprotein. Moreover, ECARs were dependent on the metabolic state of the cell and were (2.8 +/- 1.2) x 10(6) and (8.0 +/- 1.5) x 10(6) protons/cell/s in glucose-deficient and glucose-fed NIH-MDR-G185 cells, respectively, after verapamil (10 muM) stimulation. The ECARs were practically identical to the rates of lactate extrusion and thus reflect the rates of ATP synthesis via glycolysis. Taking into account the number of P-glycoprotein molecules per cell, the rate of ATP hydrolysis in inside-out vesicles of the same cells was determined as (9.2 +/- 1.5) x 10(6) phosphates/cell/s, in good agreement with the rate of ATP synthesized in glucose-fed cells. The energy required for P-glycoprotein activation relative to the basal metabolic energy was twice as large in glucose-deficient as in glucose-fed cells, suggesting cellular protection by P-glycoprotein even under conditions of starvation

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-G185 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) microM). Moreover we determined the free energy of drug binding from water to the activating binding region of the transporter (DeltaG degrees (tw(1)) = (-30 to -54) kJ/mol), the free energy of drug partitioning into the lipid membrane (DeltaG 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 (DeltaG degrees (tl(1)) = (-7 to -27) kJ/mol). For the compounds tested DeltaG degrees (tl(1)) was less negative than DeltaG degrees (lw) but varied more strongly. The free energies of substrate binding to the transporter within the lipid phase, DeltaG degrees (tl(1)), are consistent with a modular binding concept, where the energetically most efficient binding module comprises two hydrogen bond acceptor groups

P-glycoprotein senses its substrates and the lateral membrane packing density : consequences for the catalytic cycle

P-glycoprotein (ABCB1) prevents absorption (e.g., blood-brain barrier) or enhances excretion (e.g., kidney) by moving substrates from the cytosolic to the extracellular membrane leaflet at the expense of ATP hydrolysis. It translocates various drugs and functions in membranes exhibiting different lateral packing densities. To gain more functional insight, we measured the temperature dependence of the P-glycoprotein ATPase activity in NIH-MDR1-G185 cell membranes in the absence and presence of three drugs (promazine, verapamil, and PSC833), exhibiting significantly different transporter affinities. Activation enthalpies (Delta H(++)) and entropies ( TDelta S(++)) were derived from Eyring plots. In the absence of drugs, the activation enthalpy and the free energy of activation for P-glycoprotein ATPase activity was determined as Delta H(++) = 92.6 +/- 4.2 kJ/mol and Delta G(++) = 73.1 +/- 7.2 kJ/mol, respectively. Increasing the drug concentration reduced the activation enthalpy, whereby the drug with the highest transporter affinity had the strongest effect (DeltaDelta H(++) = -21%). The free energy of activation decreased for activating (DeltaDelta G(++) = approximately -3.8%) and increased for inhibitory compounds (DeltaDelta G(++) = approximately +0.7%). The drug-specific changes of the free energy of activation are thus barely above thermal energy. A comparison with literature data revealed that a decrease of the lateral membrane packing density reduces the enthalpic and the entropic contribution to the free energy of activation. Although the P-glycoprotein ATPase activity increases only slightly with decreasing lateral membrane packing density, the mode of action changes from strongly entropy-driven at high, to essentially enthalpy-driven at low packing densities. This suggests that the transporter and the membrane form a functional entity

Interaction of Verapamil with Lipid Membranes and P-Glycoprotein: Connecting Thermodynamics and Membrane Structure with Functional Activity

Verapamil and amlodipine are calcium ion influx inhibitors of wide clinical use. They are partially charged at neutral pH and exhibit amphiphilic properties. The noncharged species can easily cross the lipid membrane. We have measured with solid-state NMR the structural changes induced by verapamil upon incorporation into phospholipid bilayers and have compared them with earlier data on amlodipine and nimodipine. Verapamil and amlodipine produce a rotation of the phosphocholine headgroup away from the membrane surface and a disordering of the fatty acid chains. We have determined the thermodynamics of verapamil partitioning into neutral and negatively charged membranes with isothermal titration calorimetry. Verapamil undergoes a pK-shift of ΔpK(a) = 1.2 units in neutral lipid membranes and the percentage of the noncharged species increases from 5% to 45%. Verapamil partitioning is increased for negatively charged membranes and the binding isotherms are strongly affected by the salt concentration. The electrostatic screening can be explained with the Gouy-Chapman theory. Using a functional phosphate assay we have measured the affinity of verapamil, amlodipine, and nimodipine for P-glycoprotein, and have calculated the free energy of drug binding from the aqueous phase to the active center of P-glycoprotein in the lipid phase. By combining the latter results with the lipid partitioning data it was possible, for the first time, to determine the true affinity of the three drugs for the P-glycoprotein active center if the reaction takes place exclusively in the lipid matrix

P-glycoprotein substrate transport assessed by comparing cellular and vesicular ATPase activity

We compared the ATPase activity in inside-out vesicles and living cells with the aim to detect substrate transport using six substrates which differ significantly in their passive influx through the plasma membrane. In cells, the cytosolic membrane leaflet harboring the substrate binding site of P-glycoprotein has to be approached by passive diffusion through the lipid membrane, whereas in inside-out plasma membrane vesicles, it is accessible directly from the aqueous phase. Compounds exhibiting fast passive influx compared to active efflux by P-glycoprotein induced similar ATPase activity profiles in cells and inside-out plasma membrane vesicles, because the substrate concentrations in the cytosolic leaflet of the two systems were similar. Compounds exhibiting similar influx as efflux induced in contrast different ATPase activity profiles in cells and inside-out vesicles, because the substrate concentration in the cytosolic leaflet was significantly lower in cells than in inside-out vesicles because Pgp could cope with influx. Data show that Pgp transported all compounds at a rate proportional to ATP hydrolysis (i.e. all compounds were substrates). But entry into the cell was prevented only if passive influx of substrates across the lipid bilayer was in a similar range as active efflux of Pgp

The role of size and charge for blood-brain barrier permeation of drugs and fatty acids

The lipid bilayer is the diffusion barrier of biological membranes. Highly protective membranes such as the blood-brain barrier (BBB) are reinforced by ABC transporters such as P-glycoprotein (MDR1, ABCB1) and multidrug resistance associated proteins (MRPs, ABCCs). The transporters bind their substrates in the cytosolic lipid bilayer leaflet before they reach the cytosol and flip them to the outer leaflet. The large majority of drugs targeted to the central nervous system (CNS) are intrinsic substrates of these transporters. Whether an intrinsic substrate can cross the BBB depends on whether passive influx is higher than active efflux. In this paper, we show that passive influx can be estimated quantitatively on the basis of Stokesian diffusion, taking into account the ionization constant and the cross-sectional area of the molecule in its membrane bond conformation, as well as the lateral packing density of the membrane. Active efflux by ABC transporters was measured. The calculated net flux is in excellent agreement with experimental results. The approach is exemplified with several drugs and fatty acid analogs. It shows that compounds with small cross-sectional areas (A(D) > 70 A(2)) and/or intermediate or low charge exhibit higher passive influx than efflux and, therefore, cross the BBB despite being intrinsic substrates. Large (A(D) < 70 A(2)) or highly charged compounds show higher efflux than influx. They cannot cross the BBB and are, thus, apparent substrates for ABC transporters. The strict size and charge limitation for BBB permeation results from the synergistic interaction between passive influx and active efflux