Vol. 61, Issue 6, 1366-1376, June 2002

Characterization of the Dexniguldipine Binding Site in the Multidrug Resistance-Related Transport Protein P-Glycoprotein by Photoaffinity Labeling and Mass Spectrometry

Christoph Borchers,¹ Rainer Boer, Kurt Klemm, Volker Figala, Thomas Denzinger, Wolf-Rüdiger Ulrich, Sabine Haas, Wolfgang Ise, Volker Gekeler, and Michael Przybylski

Department of Chemistry, Analytical Chemistry, University of Konstanz, Konstanz, Germany (C.B., T.D., M.P.); and Byk Gulden Lomberg GmbH, Konstanz, Germany (R.B., K.K., V.F., W.-R.U., S.H., W.I., V.G.)

Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [³H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [³H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468-527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.