Elsevier

Biochemical Pharmacology

Volume 58, Issue 9, 1 November 1999, Pages 1447-1456
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Structure–activity relationship studies of propafenone analogs based on P-glycoprotein ATPase activity measurements

https://doi.org/10.1016/S0006-2952(99)00229-4Get rights and content

Abstract

Propafenone analogs (PAs) were previously identified as potent inhibitors of P-glycoprotein (Pgp)-mediated toxin efflux. For this as well as other classes of Pgp inhibitors, lipophilicity as well as hydrogen bond acceptor strength are important determinants of biological activity. The question as to whether a direct interaction between PA-type modulators and Pgp takes place was addressed by means of Pgp ATPase measurements and transport studies. Propafenone-type modulators stimulated ATPase activity up to 2-fold over basal activity in a concentration-dependent biphasic manner. Within a series of structural homologs, Ka values of ATPase stimulation strongly correlated with lipophilicity. Analogs containing a quaternary nitrogen stimulated Pgp ATPase activity with lesser efficacy, while Ka values were somewhat higher when compared to corresponding tertiary analogs. Transport studies performed in inside-out plasma membrane (I/O) vesicles demonstrated that analogs containing a tertiary nitrogen rapidly associated with the biomembrane. Quaternary analogs, which are restricted by a permanent positive charge in transiting the plasma membrane by diffusion, accumulated in Pgp containing I/O vesicles in an ATP-dependent and cyclosporin A-inhibitable manner, which identified them as Pgp substrates. Identical structure–activity relationships were found in either Pgp ATPase stimulation experiments in I/O vesicles or in toxin efflux inhibition studies using intact cells. Therefore, differences in membrane transit are not responsible for the observed structure–activity relationships.

Section snippets

Cell culture

The T-lymphoblast cell line CCRF-CEM and the doxorubicin-resistant subline CCRF ADR5000 were a gift from V. Gekeler (Byk Gulden, Konstanz, Germany). The cell lines have been characterized previously. For ADR5000 cells, Pgp expression has been shown to be the cause of their multidrug-resistant phenotype 10, 11, 12, 13. Cells were maintained under standard cell culture conditions. The resistant subline was cultured in the presence of 5 μg/mL doxorubicin. One week prior to the experiments, the

Effect of propafenones on P-glycoprotein ATPase activity

In the presence of ouabain, EGTA, and Na-azide, which are potent inhibitors of the membrane-bound Na/K-, Ca2+-, and mitochondrial ATPases, respectively, the Pgp-containing vesicles exhibited an ATPase activity of 39.3 ± 5 nmol/min/mg protein, which was approximately twofold higher than the activity in wt membranes. This difference is termed basal Pgp ATPase activity according to Garrigos et al. [20] and represents the sum of unstimulated Pgp ATPase and residual ATPase activities of unknown

Discussion

Measurement of Pgp ATPase activity as a function of modulator concentration is a means to obtain quantitative data describing interaction between these substances and Pgp [20]. In such assays, there is no need to simultaneously use a transported toxin such as daunorubicin or vinblastine. Potential Pgp-independent interferences between toxin and modulator, such as fluorescence quench phenomena or membrane displacement, are thus eliminated.

In the present study, propafenone analogs were shown to

Acknowledgements

This work was supported by grants from the Austrian Science Fund (P11760MOB) and from the Austrian National Bank (6899). We thank V. Gekeler (Byk Gulden, Konstanz, Germany) for providing the cell lines used in this study. We are indebted to A. Ellinger (Institute of Histology II, University of Vienna, Austria) for preparing electron micrographs of the plasma membrane preparations.

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