Abstract
P-glycoprotein (P-gp), an ATP-dependent drug efflux pump, has been implicated in multidrug resistance of several cancers as a result of its overexpression. In this work, rationally designed second-generation P-gp inhibitors are disclosed, based on dimerized versions of the substrates quinine and quinidine. These dimeric agents include reversible tethers with a built-in clearance mechanism. The designed agents were potent inhibitors of rhodamine 123 efflux in cultured cancer cell lines that display high levels of P-gp expression at the cell surface and in transfected cells expressing P-gp. The quinine homodimer Q2, which was tethered by reversible ester bonds, was particularly potent (IC50 ≈ 1.7 μM). Further studies revealed that Q2 inhibited the efflux of a range of fluorescent substrates (rhodamine 123, doxorubicin, mitoxantrone, and BODIPY-FL-prazosin) from MCF-7/DX1 cells. The reversibility of the tether was confirmed in experiments showing that Q2 was readily hydrolyzed by esterases in vitro (t½ ≈ 20 h) while demonstrating high resistance to nonenzymatic hydrolysis in cell culture media (t½ ≈ 21 days). Specific inhibition of [125I]iodoarylazidoprazosin binding to P-gp by Q2 verified that the bivalent agent interacted specifically with the drug binding site(s) of P-gp. Q2 was also an inhibitor of verapamil-stimulated ATPase activity. In addition, low concentrations of Q2 stimulated basal P-gp ATPase levels. Finally, Q2 was shown to inhibit the transport of radiolabeled paclitaxel (Taxol) in MCF-7/DX1 cells, and it completely reversed the P-gp-mediated paclitaxel resistance phenotype.
Footnotes
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The work was supported by National Institutes of Health grant EY018481 and the Purdue Research Foundation. Microscopy data were acquired in the Purdue Cancer Center Analytical Cytometry Laboratories supported by the Cancer Center NCI core grant 2P30-CA23168.
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ABBREVIATIONS: P-gp, P-glycoprotein; ABC, ATP-binding cassette; DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride; IAAP, iodoarylazidoprazosin; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HPLC, high-performance liquid chromatography; R123, rhodamine 123.
- Received July 11, 2008.
- Accepted October 22, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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