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The human P-glycoprotein (Pgp) functions as an ATP-dependent efflux pump for a large spectrum of chemically diverse therapeutic agents, including many anticancer and antimicrobial drugs. A number of compounds, termed modulators or reversing agents, with inhibitory action on Pgp-mediated drug transport have been identified, however, a need for modulators with improved clinical effectiveness is growing. A strategic approach to develop such compounds will need a clear understanding on the mode of action of the pump and the mechanisms by which its function can be modulated. We have recently demonstrated that thioxanthene-based Pgp modulators cis- and trans- flupentixols have opposite effects on ATP hydrolysis and substrate binding, in which a phenylalanine residue at position 983 (F983) plays a crucial role. To understand the mode of interaction of these modulators with Pgp, we have substituted F983 with amino acid residues containing side chains of different physicochemical properties, such as alanine (F983A), histidine (F983H), leucine (F983L), methionine (F983M), tryptophan (F983W), and tyrosine (F983Y), and studied the modulatory effects of 10 different structural analogs of flupentixol. Results indicate that residue F983 has distinct contributions in modulating ATP hydrolysis and in influencing substrate binding. All the substitutions affect the apparent Km for cis(Z)flupentixol in stimulating ATP hydrolysis, to a different extent, suggesting a role of F983 in binding of the modulator by Pgp. On other hand, stimulation of IAAP binding by cis(Z)flupentixol is completely abrogated in the mutants, which cannot be overcome by increasing the concentration of the modulator, indicating a role for F983 beyond the step of modulator binding. Interestingly, the mutations have diverse effect on inhibition of ATP hydrolysis by trans(E)flupentixol. In F983A, it inhibited IAAP binding but at a much higher concentration, while in F983M, and in F983L, ATP hydrolysis was clearly stimulated by trans(E)flupentixol, with no clear cut inhibition or stimulation in F983W and F983H, suggesting that the residue F983 may act as a molecular switch, capable of both turning on and off ATP hydrolysis, depending on the modulator structure. In contrast, all the mutants show inhibition of IAAP binding by trans(E)flupentixol but at much higher concentration, suggesting a role of the residue in binding of the modulator. Interestingly, the effect of the substitutions of the functional groups within flupentixol varies depending on the nature of the side-chain present at position 983, suggesting a direct physical interaction at the molecular level between the thioxanthene-based modulators and the residue F983.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]