Cooperativity in the inhibition of P-glycoprotein-mediated daunorubicin transport: evidence for half-of-the-sites reactivity.

P-Glycoprotein (Pgp) is an important transport enzyme composed of two homologous domains and transports a wide range of structurally diverse xenobiotics from the cell. Recent studies have indicated that allosteric interactions occur between the nucleotide binding domains and between the substrate binding domains of the two halves, but the extent of this interaction as well as the means by which the enzyme can transport such a wide variety of substrates has not been elucidated. Herein, the Pgp-mediated transport of a marker substrate, daunorubicin (DNR), out of viable cells was examined in the presence of a variety of other known substrates of Pgp. For most of the typical Pgp substrates examined, the relationship between inhibition of DNR efflux and competing substrate concentration was sigmoidal and therefore not a simple mutually exclusive competitive inhibition of transport. The Hill coefficient ranged from about 3 to 5 for the inhibition of transport of DNR. This negative cooperativity in combination with recent evidence, including several examples of noncompetitive inhibition between the homologous halves of Pgp, indicates a "half-of-the-sites" reactivity. Our data support the mechanistic proposal that substrate binding at one putative transport binding site precludes activity at another unequal site; many of the substrates examined exert a negative allosteric effect on the other transport site (and vice versa). A half-of-the-sites reactivity model would account for many of these observations and may be critical to the efficiency of Pgp substrate transport of a broad spectrum of compounds.