P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure function relationships

Resistance to chemotherapy in cancer cells is mainly mediated by overexpres sion of P-glycoprotein (Pgp), a plasma membrane ATP-binding cassette (ABC) transporter which extrudes cytotoxic drugs at the expense of ATP hydrolysis . Pgp consists of two homologous halves each containing a transmembrane dom ain and a cytosolic nucleotide-binding domain (NBD) which contains two cons ensus Walker motifs, A and B, involved in ATP binding and hydrolysis, The p rotein also contains an S signature characteristic of ABC transporters, The molecular mechanism of Pgp-mediated drug transport is not known. Since the transporter has an extraordinarily broad substrate specificity, its cellul ar function has been described as a "hydrophobic vacuum cleaner". The limit ed knowledge about the mechanism of Pgp, partly due to the lack of a high-r esolution structure, is well reflected in the failure to efficiently inhibi t its activity in cancer cells and thus to reverse multidrug resistance (MD R), In contrast to the difficulties encountered when studying the full-leng th Pgp, the recombinant NBDs can be obtained in large amounts as soluble pr oteins. The biochemical and biophysical characterization of recombinant NBD s is shown here to provide a suitable alternative route to establish struct ure-function relationships. NBDs were shown to bind ATP and analogues as we ll as potent modulators of MDR, such as hydrophobic steroids, at a region c lose to the ATP site. Interestingly, flavonoids also bind to NBDs with high affinity. Their binding site partly overlaps both the ATP-binding site and the steroid-interacting region. Therefore flavonoids constitute a new prom ising class of bifunctional modulators of Pgp.