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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, [3 H]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 [3 H]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.Tumor cells in vitro and in vivo can develop simultaneous resistance to the lethal effects of a variety of cytotoxic drugs (Endicott and Ling, 1989). This so-called multidrug resistance (MDR) is a major limiting factor for the efficacy of cancer chemotherapy. Currently, a variety of mechanisms are known that can lead to drug resistance, including reduced cellular drug accumulation, increased detoxification, intracellular vesicularization of drugs, altered enzymatic activities, up-or down-regulation of targets, and enhanced DNA repair (Hayes and Wolf, 1990). One important resistance mechanism is the transport out of cancer cells of chemically-unrelated cytotoxic drugs (such as anthracylines, Vinca alkaloids, colchicine, and taxanes) by the integral membrane phosphoglycoprotein P-glycoprotein (P-gp) under ATP hydrolysis, resulting in low and ineffective intracellular drug concentrations (Gottesman and Pastan, 1993). Although considerable progress has been made during the last few years, the mechanism of recognition and transport of such a broad spectrum of compounds is still poorly understood.Reversal of multidrug resistance is of major clinical interest, and MDR-reversing agents called chemosensitizers have been intensively investigated (Raderer and Scheithauer, 1993;Sikic, 1993). The capability of reversi...

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The (H+-K+)-ATPase inhibiting sulfoxides la,b undergo acid-induced transformations into the cyclic sulfenamides 2a,b via the putative sulfenic acids 4a,b. At high concentrations mixtures of 2a,b and thiosulfinates 5a,b were obtained. In contrast, IC, which cannot form 2, is completely transformed under similar conditions into 5c, the anhydride of 4c. The temperature-dependent equilibrium between 2a and 5a and the kinetics of the ring opening of 2a by water provide evidence for the sulfenic acid 4 as an intermediate involved in the pH-dependent transformations of 1. The thiossulfnates 5 decompose mainly to the disulfides 6 at a rate depending on the solvent and conditions and are also reduced to 6 by thiols. The dimeric ylide 7a could be isolated from 6a (prepared in situ from la as well as from 2a). The symmetrical disulfides 6 and 7 are cleaved by thiols to yield the mixed disulfides 3 and the thiols 8. Similarly, cleavage of 3 liberates an additional equivalent of 8. Finally, the unstable thiols 8 are removed from the thiol-disulfide interchange equilibrium by irreversible rearrangement to the sulfides 9.