Synthesis, Pharmacologic Activity, and Structure-Activity Relationships of a Series of Propafenone-Related Modulators of Multidrug Resistance

Chiba, Peter; Burghofer, Sabine; Richter, Ernst-Jürgen; Tell, Barbara; Moser, A.; Ecker, Gerhard

doi:10.1021/jm00014a031

Cited by 61 publications

(56 citation statements)

References 7 publications

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“…However, similar to quaternary compounds, propafenone analogs containing a tertiary nitrogen atom caused a shift in fluorescence when P-gp was reacted with the conformation-sensitive antibody UIC2 (S. Park and P. Chiba, unpublished observations). In addition, a number of studies from our group demonstrated that propafenones inhibit the transport of known P-gp substrates, including rhodamine 123, daunorubicin (Chiba et al, 1995;Ecker et al, 1999Ecker et al, , 2002, mitoxantrone, and vinblastine (S. Kopp and P. Chiba, unpublished observations). Photolabeling of plasma-membrane vesicles with the radioactive analog […”

Section: Resultsmentioning

confidence: 93%

P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

Pleban¹,

Kopp²,

Csaszar³

et al. 2004

Mol Pharmacol

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121

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P-glycoprotein (P-gp) is an energy-dependent multidrug efflux pump conferring resistance to cancer chemotherapy. Characterization of the mechanism of drug transport at a molecular level represents an important prerequisite for the design of pump inhibitors, which resensitize cancer cells to standard chemotherapy. In addition, P-glycoprotein plays an important role for early absorption, distribution, metabolism, excretion, and toxicity profiling in drug development. A set of propafenonetype substrate photoaffinity ligands has been used in this study in conjunction with matrix-assisted laser desorption/ionization timeof-flight mass spectrometry to define the substrate binding domain(s) of P-gp in more detail. The highest labeling was observed in transmembrane segments 3, 5, 8, and 11. A homology model for P-gp was generated on the basis of the dimeric crystal structure of Vibrio cholerae MsbA, an essential lipid transporter. Thereafter, the labeling pattern was projected onto the 3D atomic-detail model of P-gp to allow a visualization of the binding domain(s). Labeling is predicted by the model to occur at the two transmembrane domain/transmembrane domain interfaces formed between the amino-and carboxyl-terminal half of P-gp. These interfaces are formed by transmembrane (TM) segments 3 and 11 on one hand and TM segments 5 and 8 on the other hand. Available data on LmrA and AcrB, two bacterial multidrug efflux pumps, suggest that binding at domain interfaces may be a general feature of polyspecific drug efflux pumps.Multidrug resistance represents a serious obstacle to successful cancer chemotherapy. Although multifactorial in etiology, one type of multidrug resistance is associated with the overexpression of energy-dependent membrane-bound pumps, which intercept and efflux drugs before they reach their intracellular target structures. P-glycoprotein (ABCB1) represents a paradigm ATP-dependent efflux pump expressed in human cancer cells. In addition to its expression in cancer cells, P-gp is also physiologically expressed in a number of tissues such as intestinal epithelial cells, at the brush border of renal tubule epithelial cells, the canalicular side of hepatocytes, and in capillary endothelial cells forming the blood-brain barrier. It thus interferes with oral drug absorption and drug delivery to the brain, and it enhances renal and biliary excretion. P-gp has therefore attracted considerable attention as a nontarget in the field of drug development, because for a large number of active compounds, interaction with P-glycoprotein might compromise their future development into a drug. Considerable energy has therefore been devoted to the characterization of molecular features that make compounds P-gp substrates and to the definition of the molecular mechanism of drug transport by P-gp. A number of studies have dealt with the kinetics and thermodynamics of the transport process

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Section: Resultsmentioning

confidence: 93%

P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

Pleban¹,

Kopp²,

Csaszar³

et al. 2004

Mol Pharmacol

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121

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“…The permanently charged ammonium derivative GPV708 was obtained via methylation of the corresponding diethylamino-derivative (Chiba 1995). IR, MS, NMR spectroscopy and elemental analysis confirmed the structure of all novel compounds.…”

Section: Methodsmentioning

confidence: 85%

“…Synthesis of compounds GPV05 to GPV180 was achieved as described previously (Chiba et al 1995Tmej et al, 1998). Benzophenone derivatives were synthesized in analogy to the procedures for GPV317 and GPV319 as outlined in Scheme 1 (Tmej et al, 1998).…”

Section: Methodsmentioning

confidence: 99%

Identification of Ligand-Binding Regions of P-Glycoprotein by Activated-Pharmacophore Photoaffinity Labeling and Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry

et al. 2002

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Energy dependent efflux pumps confer resistance to anticancer, antimicrobial, and antiparasitic drugs. P-glycoprotein (Pgp, ABCB1) mediates resistance to a broad spectrum of antitumor drugs. Compounds that themselves are nontoxic to cells have been shown to act as inhibitors of Pgp. The mechanism of binding and transport of low-molecular-mass ligands by Pgp is still incompletely understood. This study introduces a series of propafenone-related photoaffinity ligands, which combine high specificity and selectivity for Pgp with high labeling efficiency. Molecules are intrinsically photoactivatable in the arylcarbonyl group, which represents a pharmacophoric substructure for this group of ligand molecules. A detailed study of the structure-activity relationship for this type of photoligand is presented. In subsequent experiments, these ligands were used to characterize the drug-binding domain of propafenone-type analogs. Matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry shows that propafenone-type ligands preferentially label fragments assigned to putative transmembrane segments 3, 5, 6, 8, 10, 11, and 12. Labeled fragments are also identified in a highly charged region of 15 amino acids in the second cytoplasmic loop. This region corresponds to the so-called EAA-like motif, which has been proposed to play a role in the interaction between transmembrane domain and nucleotide binding domain of peroxisomal ATP-binding cassette transporters. In addition, a region in cytoplasmic loop 3 and between TM12 and the N terminus of the Walker A sequence of NBD2 are labeled by the ligands. Therefore, a number of confined protein regions contribute to the drug-binding domain of propafenone-type analogs.

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“…The design and synthesis of propafenone-type compounds including the photo-affinity ligands has been described previously (Chiba et al, 1995(Chiba et al, , 1996Ecker et al, 2002). Structures were confirmed by IR, mass spectroscopy, NMR spectroscopy, and elemental analysis.…”

Section: Methodsmentioning

confidence: 99%

A Three-Dimensional Model for the Substrate Binding Domain of the Multidrug ATP Binding Cassette Transporter LmrA

Ecker

Pleban²,

Kopp³

et al. 2004

Mol Pharmacol

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Multidrug resistance presents a major obstacle to the treatment of infectious diseases and cancer. LmrA, a bacterial ATP-dependent multidrug transporter, mediates efflux of hydrophobic cationic substrates, including antibiotics. The substrate-binding domain of LmrA was identified by using photo-affinity ligands, proteolytic degradation of LmrA, and identification of ligand-modified peptide fragments with matrix-assisted laser desorption ionization/time of flight mass spectrometry. In the nonenergized state, labeling occurred in the ␣-helical transmembrane segments (TM) 3, 5 and 6 of the membrane-spanning domain. Upon nucleotide binding, the accessibility of TM5 for substrates increased, whereas that of TM6 decreased. Inverse changes were observed upon ATP-hydrolysis. An atomic-detail model of dimeric LmrA was generated based on the template structure of the homologous transporter MsbA from Vibrio cholerae, allowing a three-dimensional visualization of the substrate-binding domain. Labeling of TM3 of one monomer occurred in a predicted area of contact with TM5 or TM6 of the opposite monomer, indicating substrate-binding at the monomer/monomer interface. Inverse changes in the reactivity of TM segments 5 and 6 suggest that substrate binding and release involves a repositioning of these helices during the catalytic cycle.

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Synthesis, Pharmacologic Activity, and Structure-Activity Relationships of a Series of Propafenone-Related Modulators of Multidrug Resistance

Cited by 61 publications

References 7 publications

P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

P-Glycoprotein Substrate Binding Domains Are Located at the Transmembrane Domain/Transmembrane Domain Interfaces: A Combined Photoaffinity Labeling-Protein Homology Modeling Approach

Identification of Ligand-Binding Regions of P-Glycoprotein by Activated-Pharmacophore Photoaffinity Labeling and Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry

A Three-Dimensional Model for the Substrate Binding Domain of the Multidrug ATP Binding Cassette Transporter LmrA

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