A Novel Electron Paramagnetic Resonance Approach to Determine the Mechanism of Drug Transport by P-glycoprotein

Abstract

ATP-driven pumping of a variety of drugs out of cells by the human P-glycoprotein poses a serious problem to medical therapy. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Membrane permeability of transported drugs and consequent lack of an experimentally defined drug position have made resolution of the transport mechanism difficult by classical techniques. To overcome these obstacles we devised a novel EPR spin-labeled verapamil for use as a transport substrate. Spin-labeled verapamil was an excellent transport substrate with apparent turnover number, K m and K i values of 5.8 s ؊1 , 4 M, and 210 M, respectively, at pH 7.4 and 37°C. The apparent affinities were ϳ10-fold higher than for unlabeled verapamil. Spin-labeled verapamil stimulated ATPase activity ϳ5-fold, was relatively hydrophilic, and had a very low flip-flop rate, making it an ideal transport substrate. The K m for MgATP activation of transport was 0.8 mM. By measuring the mobility of spin-labeled verapamil during transport experiments, we were able to resolve the location of the drug in proteoliposome suspensions. Steady state gradients of spin-labeled verapamil within the range of K i /K m ratios were observed.

Human P-glycoprotein Contains a Greasy Ball-and-Socket Joint at the Second Transmission Interface

“…The histidine-tagged mutants were expressed in HEK 293 cells, isolated by nickel-chelate chromatography, and assayed for verapamil-stimulated ATPase activity. Verapamil was used because it is transported by P-gp (35) and it highly stimulates the ATPase activity of human P-gp (over 10-fold) (36). It was found that the activity of all three mutants was similar to that of wild-type P-gp (Fig.…”

“…P-gp can be trapped at a transition state during ATP hydrolysis by including vanadate in the reaction mix (35). Vanadate traps ADP at either NBD by mimicking the transition state of the ␥-phosphate of ATP during hydrolysis, and trapping at one NBD inhibits ATP hydrolysis at the second site (35). Vanadate trapping of nucleotide causes conformational changes in P-gp, and this can be detected by disulfide cross-linking analysis (36).…”

“…To test whether mutant F1086A retained the ability to bind and hydrolyze ATP, we tested whether vanadate trapping of ATP would still inhibit cross-linking of mutant F1086A/L443C/S909C. P-gp can be trapped at a transition state during ATP hydrolysis by including vanadate in the reaction mix (35). Vanadate traps ADP at either NBD by mimicking the transition state of the ␥-phosphate of ATP during hydrolysis, and trapping at one NBD inhibits ATP hydrolysis at the second site (35).…”

Physiological concentrations of albumin favor drug binding

“…Spin labeled drugs have been synthesized and used to study the drug mechanisms at a molecular level for a long time. [11][12][13] Besides their reporter properties, nitroxides are also used in anti-oxidant therapies for several diseases because they can effectively scavenge the reactive oxygen species and therefore improve cell viability. 14,15 Here, we use continuous wave (cw) EPR spectroscopy as an alternative technique to study the interaction between drugs and proteins.…”

“…One major advantage of cw EPR spectroscopy is the ability to observe the unbound drug and the bound drug simultaneously, due to its sensitivity to the rotational dynamics of the radicals. [11][12][13] Bound spin labeled molecules (immobilized) have characteristic broad signals stemming from restricted rotational motion, and unbound spin labeled molecules (mobilized) have sharp three-line signals coming from freely tumbling motion. [16][17][18] Salicylic acid (SA), which is a non-steroidal anti-inflammatory drug, and serum albumin, which is the most abundant plasma protein, were used as a model drug and transporter protein, respectively.…”