Ligand-mediated tertiary structure changes of reconstituted P-glycoprotein- A tryptophan fluorescence quenching analysis

Ligand-dependent changes in accessibility of purified P-glycoprotein, funct ionally reconstituted in liposomes, were investigated by fluorescence measu rements.. Trp quenching experiments provided evidence that P-glyeoprotein a dopts different tertiary structures upon binding of drug substrates in the absence and presence of MgATP and its nonhydrolyzable analog, MgATP gamma S , Five anthracycline derivatives were tested as drug substrates: daunorubic in, 4'-epi-doxorubicin, iododoxorubicin, 4-demethoxy-daunorubicin, and meth oxy-morpholino-doxorubicin. Among them, daunorubicin and 4'-epi-doxorubicin have been shown to be rejected outside the multidrug-resistant cells, wher eas the three others have been shown to accumulate in multidrug-resistant c ells overexpressing P-glycoprotein and therefore retain their cytotoxic act ivity. A small conformational change was associated with nucleotide 'bindin g and amplified after nucleotide hydrolysis, Different conformational state s were adopted by P-glycoprotein upon the addition of the anthracycline der ivatives in the absence and presence of MgATP or MgATP gamma S. These confo rmational changes are shown to be related to the nature of the antitumor ag ents and more precisely to their capacity to accumulate in resistant cells, These data also suggest that the cytotoxicity of iododoxorubicin and 4-dem ethoxy-daunorubicin is related to the fact they are not transported by P-gl ycoprotein, On the contrary, methoxy-morpholino-doxorubicin cytotoxicity ma y be explained in terms of its rapid reincorporation into the:plasma membra ne after being transported by P-glycoprotein.