Abstract
The aim of the present study was to demonstrate that the modulation of P-glycoprotein (Pgp) ATPase activity by peptides, drugs, and chemosensitizers takes place on a common drug pharmacophore. To this end, a highly emetine-resistant Chinese hamster ovary cell line was established, in which Pgp constituted 18% of plasma membrane protein. Reconstituted proteoliposomes, the Pgp content of which was up to 40%, displayed a basal activity of 2.6 +/- 0.45 micromol of Pi/min/mg of protein, suggesting the presence of an endogenous Pgp substrate. This basal ATPase activity was stimulated (up to 5.2 micromol of Pi/min/mg of protein) by valinomycin and various Pgp substrates, whereas, to our surprise, gramicidin D, an established Pgp substrate, was inhibitory. Taking advantage of this novel inhibition of Pgp ATPase activity by gramicidin D, a drug competition assay was devised in which gramicidin D-inhibited Pgp ATPase was coincubated with increasing concentrations of various substrates that stimulate its ATPase activity. Gramicidin D inhibition of Pgp ATPase was reversed by Pgp substrates, including various cytotoxic agents and chemosensitizers. The inhibition of the basal ATPase activity and the reversal of gramicidin D inhibition of Pgp ATPase by its various substrates conformed to classical Michaelis-Menten competition. This competition involved an endogenous substrate, the inhibitory drug gramicidin D, and a stimulatory substrate. We conclude that the various MDR type substrates and chemosensitizers compete on a common drug binding site present in Pgp.
Highlights
Inherent as well as acquired antitumor drug resistance continue to pose major obstacles toward the successful chemotherapeutic treatment of various human malignancies [1]
Using Pgp-reconstituted proteoliposomes, we have recently shown an ATP-driven transport of valinomycin and gramicidin D [15]; this active intravesicular accumulation of peptide ionophores was blocked by established Pgp substrates including the anthracyclines doxorubicin, daunorubicin, the Vinca alkaloid vinblastine as well as the tripeptide N-acetylleucyl-leucyl-norleucinal (ALLN; see Ref. 32)
Using proteoliposomes reconstituted with Pgp ATPase, we show here that various hydrophobic peptides, cytotoxic drugs, and MDR chemosensitizers compete on a common drug pharmacophore present in Pgp
Summary
Inherent as well as acquired antitumor drug resistance continue to pose major obstacles toward the successful chemotherapeutic treatment of various human malignancies [1]. Sharom et al [14] showed an ATP-dependent transport of colchicine into proteoliposomes reconstituted with hamster Pgp, whereas Eytan et al [15] demonstrated an ATPdriven transport of the hydrophobic peptide ionophores gramicidin D and valinomycin into proteoliposomes reconstituted with rat and hamster Pgp. Pgp has been shown to bind the photoaffinity ATP analogue 8-azido-ATP [16, 17], and amino acid substitutions of either or both the ATP-binding domains resulted in abolished function of drug transport. Smit et al [24], used targeted MDR1 gene disruption in transgenic mice and found these mice to be 100fold more sensitive to ivermectin, a central nervous system neurotoxin, strongly suggesting that the physiological overexpression of Pgp in the blood brain barrier endothelium [25] is presumably responsible for cytotoxins extrusion Taken together, these results strongly suggest that Pgp functions as an ATP-dependent efflux transporter of multiple hydrophobic cytotoxic drugs. Using Pgp-reconstituted proteoliposomes, we have recently shown an ATP-driven transport of valinomycin and gramicidin D [15]; this active intravesicular accumulation of peptide ionophores was blocked by established Pgp substrates including the anthracyclines doxorubicin, daunorubicin, the Vinca alkaloid vinblastine as well as the tripeptide N-acetylleucyl-leucyl-norleucinal (ALLN; see Ref. 32)
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