ACS Publications. Most Trusted. Most Cited. Most Read
My Activity

Figure 1Loading Img

Binding of Steroid Modulators to Recombinant Cytosolic Domain from Mouse P-Glycoprotein in Close Proximity to the ATP Site

View Author Information
Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, UPR 412 du Centre National de la Recherche Scientifique, Lyon, France, Institut National de la Santé et de la Recherche Médicale U 33, Le Kremlin-Bicêtre, France, and Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada
Cite this: Biochemistry 1997, 36, 49, 15208–15215
Publication Date (Web):December 9, 1997
Copyright © 1997 American Chemical Society

    Article Views





    Read OnlinePDF (236 KB)


    We recently found that recombinant NBD1 cytosolic domain corresponding to segment 395−581 of mouse mdr1 P-glycoprotein bound fluorescent 2‘(3‘)-N-methylanthraniloyl-ATP (MANT-ATP) with high affinity [Dayan, G., Baubichon-Cortay, H., Jault, J.-M., Cortay, J.-C., Deléage, G., & Di Pietro, A. (1996) J. Biol. Chem.271, 11652−11658]. The present work shows that a longer 371−705 domain (extended-NBD1), including tryptophan-696 as an intrinsic probe, which bound MANT-ATP with identical affinity, also interacted with steroids known to modulate anticancer drug efflux from P-glycoprotein-positive multidrug-resistant cells. Progesterone, which is not transported, its hydrophobic derivatives medroxyprogesterone acetate and megestrol acetate, and Δ6-progesterone produced nearly a 50% saturating quenching of the domain intrinsic fluorescence, with dissociation constants ranging from 53 to 18 μM. The even more hydrophobic antiprogestin RU 486 produced a complete quenching of tryptophan-696 fluorescence, in contrast to more hydrophilic derivatives of progesterone containing hydroxyl groups at positions 11, 16, 17, and 21 and known to be transported, which produced very little quenching. A similar differential interaction was observed with full-length purified P-glycoprotein. The steroid-binding region within extended-NBD1 appeared distinct from the nucleotide-binding site as the RU 486-induced quenching was neither prevented nor reversed by high ATP concentrations. In contrast, MANT-ATP binding was efficiently prevented or displaced by RU 486, suggesting that the hydrophobic MANT group of the bound nucleotide analogue overlaps, at least partially, the adjacent steroid-binding region revealed by RU 486.

     This work was supported by grants from the CNRS (UPR 412), the Université Claude Bernard de Lyon, and the Fondation de France (Grant 96003891).


     Institut de Biologie et Chimie des Protéines.

     Institut National de la Santé et de la Recherche Médicale U 33.

     McGill University.


     Corresponding author address:  Laboratoire de Biochimie Structurale et Fonctionnelle, Institut de Biologie et Chimie des Protéines, UPR 412 du CNRS, Université Claude Bernard-Lyon I, 7 Passage du Vercors, 69367 Lyon Cedex 07, France. Tel:  33-4 72 72 26 29. Fax:  33-4 72 72 26 01. E-mail:  [email protected].

     Abstract published in Advance ACS Abstracts, November 15, 1997.

    Cited By

    This article is cited by 49 publications.

    1. Claudia P. Alvarez, Marijana Stagljar, D. Ranjith Muhandiram, and Voula Kanelis . Hyperinsulinism-Causing Mutations Cause Multiple Molecular Defects in SUR1 NBD1. Biochemistry 2017, 56 (18) , 2400-2416.
    2. Marc Rolland de Ravel, Ghina Alameh, Maxime Melikian, Zahia Mahiout, Agnès Emptoz-Bonneton, Eva-Laure Matera, Thierry Lomberget, Roland Barret, Luc Rocheblave, Nadia Walchshofer, Sonia Beltran, Lucienne El Jawad, Elisabeth Mappus, Catherine Grenot, Michel Pugeat, Charles Dumontet, Marc Le Borgne, and Claude Yves Cuilleron . Synthesis of New Steroidal Inhibitors of P-Glycoprotein-Mediated Multidrug Resistance and Biological Evaluation on K562/R7 Erythroleukemia Cells. Journal of Medicinal Chemistry 2015, 58 (4) , 1832-1845.
    3. Elvin D. de Araujo, Lynn K. Ikeda, Svetlana Tzvetkova, and Voula Kanelis . The First Nucleotide Binding Domain of the Sulfonylurea Receptor 2A Contains Regulatory Elements and Is Folded and Functions as an Independent Module. Biochemistry 2011, 50 (31) , 6655-6666.
    4. Paul D. W. Eckford and Frances J. Sharom. ABC Efflux Pump-Based Resistance to Chemotherapy Drugs. Chemical Reviews 2009, 109 (7) , 2989-3011.
    5. Amanda B. Chai, Anika M. S. Hartz, Xuexin Gao, Alryel Yang, Richard Callaghan, Ingrid C. Gelissen. New Evidence for P-gp-Mediated Export of Amyloid-β Peptides in Molecular, Blood-Brain Barrier and Neuronal Models. International Journal of Molecular Sciences 2021, 22 (1) , 246.
    6. Mohana Krishna Gopisetty, Dóra Izabella Adamecz, Ferenc István Nagy, Ádám Baji, Vasiliki Lathira, Márton Richárd Szabó, Renáta Gáspár, Tamás Csont, Éva Frank, Mónika Kiricsi. Androstano-arylpyrimidines: Novel small molecule inhibitors of MDR1 for sensitizing multidrug-resistant breast cancer cells. European Journal of Pharmaceutical Sciences 2021, 156 , 105587.
    7. Santiago Vilar, Eduardo Sobarzo-Sánchez, Eugenio Uriarte. In Silico Prediction of P-glycoprotein Binding: Insights from Molecular Docking Studies. Current Medicinal Chemistry 2019, 26 (10) , 1746-1760.
    8. Carmen W. Dessauer, Val J. Watts, Rennolds S. Ostrom, Marco Conti, Stefan Dove, Roland Seifert, . International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases. Pharmacological Reviews 2017, 69 (2) , 93-139.
    9. Tingting Guo, Jinhu Huang, Hongyu Zhang, Lingling Dong, Dawei Guo, Li Guo, Fang He, Zohaib Ahmed Bhutto, Liping Wang. Abcb1 in Pigs: Molecular cloning, tissues distribution, functional analysis, and its effect on pharmacokinetics of enrofloxacin. Scientific Reports 2016, 6 (1)
    10. Edna Ooko, Tahseen Alsalim, Bahjat Saeed, Mohamed E.M. Saeed, Onat Kadioglu, Hanna S. Abbo, Salam J.J. Titinchi, Thomas Efferth. Modulation of P-glycoprotein activity by novel synthetic curcumin derivatives in sensitive and multidrug-resistant T-cell acute lymphoblastic leukemia cell lines. Toxicology and Applied Pharmacology 2016, 305 , 216-233.
    11. Maen Zeino, Malte S. Paulsen, Martin Zehl, Ernst Urban, Brigitte Kopp, Thomas Efferth. Identification of new P-glycoprotein inhibitors derived from cardiotonic steroids. Biochemical Pharmacology 2015, 93 (1) , 11-24.
    12. Durg Vijay Singh, Madan M. Godbole, Krishna Misra. A plausible explanation for enhanced bioavailability of P-gp substrates in presence of piperine: simulation for next generation of P-gp inhibitors. Journal of Molecular Modeling 2013, 19 (1) , 227-238.
    13. Waël Zeinyeh, Zahia Mahiout, Sylvie Radix, Thierry Lomberget, Axel Dumoulin, Roland Barret, Catherine Grenot, Luc Rocheblave, Eva-Laure Matera, Charles Dumontet, Nadia Walchshofer. Progesterone–adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux: Design, synthesis, characterization and biological evaluation. Steroids 2012, 77 (12) , 1177-1191.
    14. S. Seddiki, T. Sahraoui, F. -Z. El Kebir. Surexpression de la glycoprotéine P humaine et reversion du phénotype MDR dans les cellules R7 issues d’une érythroleucémie. Journal Africain du Cancer / African Journal of Cancer 2012, 4 (2) , 101-107.
    15. Manzerul Bhuiyan, Sophie Petropoulos, William Gibb, Stephen G. Matthews. Sertraline Alters Multidrug Resistance Phosphoglycoprotein Activity in the Mouse Placenta and Fetal Blood–Brain Barrier. Reproductive Sciences 2012, 19 (4) , 407-415.
    16. Andreia Palmeira, M. Helena Vasconcelos, Ana Paiva, Miguel X. Fernandes, Madalena Pinto, Emília Sousa. Dual inhibitors of P-glycoprotein and tumor cell growth: (Re)discovering thioxanthones. Biochemical Pharmacology 2012, 83 (1) , 57-68.
    17. Virginia Lanzotti, Orazio Taglialatela-Scafati, Ernesto Fattorusso, Attilio Di Pietro. Plant Compounds and Derivatives as Inhibitors of Cancer Cell Multidrug Resistance. 2011, 409-450.
    18. Andreia Palmeira, Freddy Rodrigues, Emília Sousa, Madalena Pinto, M. Helena Vasconcelos, Miguel X. Fernandes. New Uses for Old Drugs: Pharmacophore‐Based Screening for the Discovery of P‐Glycoprotein Inhibitors. Chemical Biology & Drug Design 2011, 78 (1) , 57-72.
    19. Waël Zeinyeh, Ghina Alameh, Sylvie Radix, Catherine Grenot, Charles Dumontet, Nadia Walchshofer. Design, synthesis and evaluation of progesterone–adenine hybrids as bivalent inhibitors of P-glycoprotein-mediated multidrug efflux. Bioorganic & Medicinal Chemistry Letters 2010, 20 (10) , 3165-3168.
    20. Miguel R. Lugo, Frances J. Sharom. Interaction of LDS-751 with the drug-binding site of P-glycoprotein: A Trp fluorescence steady-state and lifetime study. Archives of Biochemistry and Biophysics 2009, 492 (1-2) , 17-28.
    21. Sergio Mares-Sámano, Raj Badhan, Jeffrey Penny. Identification of putative steroid-binding sites in human ABCB1 and ABCG2. European Journal of Medicinal Chemistry 2009, 44 (9) , 3601-3611.
    22. Alvaro J. Benitez, Michael J. Arrowood, Jan R. Mead. Functional characterization of the nucleotide binding domain of the Cryptosporidium parvum CpABC4 transporter: An iron–sulfur cluster transporter homolog. Molecular and Biochemical Parasitology 2009, 165 (2) , 103-110.
    23. E. Nicolle, A. Boumendjel, S. Macalou, E. Genoux, A. Ahmed-Belkacem, P.-A. Carrupt, A. Di Pietro. QSAR analysis and molecular modeling of ABCG2-specific inhibitors. Advanced Drug Delivery Reviews 2009, 61 (1) , 34-46.
    24. Ling Zhang, Yoshiaki Kanda, Drucilla J. Roberts, Jeffrey L. Ecker, Ralf Losel, Martin Wehling, John J. Peluso, James K. Pru. Expression of progesterone receptor membrane component 1 and its partner serpine 1 mRNA binding protein in uterine and placental tissues of the mouse and human. Molecular and Cellular Endocrinology 2008, 287 (1-2) , 81-89.
    25. Karima Oughlissi-Dehak, Philippe Lawton, Serge Michalet, Christine Bayet, Nicole Darbour, Mahfoud Hadj-Mahammed, Yacine A. Badjah-Hadj-Ahmed, Marie-Geneviève Dijoux-Franca, David Guilet. Sesquiterpenes from aerial parts of Ferula vesceritensis. Phytochemistry 2008, 69 (9) , 1933-1938.
    26. Frances J Sharom. ABC multidrug transporters: structure, function and role in chemoresistance. Pharmacogenomics 2008, 9 (1) , 105-127.
    27. Alicia Ponte-Sucre. Availability and applications of ATP-binding cassette (ABC) transporter blockers. Applied Microbiology and Biotechnology 2007, 76 (2) , 279-286.
    28. Philippe Lawton, Michel Pélandakis, Anne-Françoise Pétavy, Nadia Walchshofer. Overexpression, purification and characterization of a hexahistidine-tagged recombinant extended nucleotide-binding domain 1 (NBD1) of the Cryptosporidium parvum CpABC3 for rational drug design. Molecular and Biochemical Parasitology 2007, 152 (1) , 101-107.
    29. José M. Pérez-Victoria, Fernando Cortés-Selva, Adriana Parodi-Talice, Boris I. Bavchvarov, F. Javier Pérez-Victoria, Francisco Muñoz-Martínez, Mathias Maitrejean, M. Paola Costi, Denis Barron, Attilio Di Pietro, Santiago Castanys, Francisco Gamarro. Combination of Suboptimal Doses of Inhibitors Targeting Different Domains of LtrMDR1 Efficiently Overcomes Resistance of Leishmania spp. to Miltefosine by Inhibiting Drug Efflux. Antimicrobial Agents and Chemotherapy 2006, 50 (9) , 3102-3110.
    30. Francisco Muñoz-Martínez, Carolina P. Reyes, Antonio L. Pérez-Lomas, Ignacio A. Jiménez, Francisco Gamarro, Santiago Castanys. Insights into the molecular mechanism of action of Celastraceae sesquiterpenes as specific, non-transported inhibitors of human P-glycoprotein. Biochimica et Biophysica Acta (BBA) - Biomembranes 2006, 1758 (1) , 98-110.
    31. Yashang Lee, Hosup Yeo, Shwu-Huey Liu, Zaoli Jiang, Ruben M. Savizky, David J. Austin, Yung-chi Cheng. Increased Anti-P-glycoprotein Activity of Baicalein by Alkylation on the A Ring. Journal of Medicinal Chemistry 2004, 47 (22) , 5555-5566.
    32. Ian D. Kerr, Georgina Berridge, Kenneth J. Linton, Christopher F. Higgins, Richard Callaghan. Definition of the domain boundaries is critical to the expression of the nucleotide-binding domains of P-glycoprotein. European Biophysics Journal 2003, 32 (7) , 644-654.
    33. Gwenaëlle Conseil, José M. Perez-Victoria, J.Michel Renoir, André Goffeau, Attilio Di Pietro. Potent competitive inhibition of drug binding to the Saccharomyces cerevisiae ABC exporter Pdr5p by the hydrophobic estradiol-derivative RU49953. Biochimica et Biophysica Acta (BBA) - Biomembranes 2003, 1614 (2) , 131-134.
    34. Georgina Berridge, Jennifer A. Walker, Richard Callaghan, Ian D. Kerr. The nucleotide‐binding domains of P‐glycoprotein. European Journal of Biochemistry 2003, 270 (7) , 1483-1492.
    35. Jie Cai, Philippe Gros. Overexpression, purification, and functional characterization of ATP-binding cassette transporters in the yeast, Pichia pastoris. Biochimica et Biophysica Acta (BBA) - Biomembranes 2003, 1610 (1) , 63-76.
    36. Ahcène Boumendjel, Attilio Di Pietro, Charles Dumontet, Denis Barron. Recent advances in the discovery of flavonoids and analogs with high‐affinity binding to P‐glycoprotein responsible for cancer cell multidrug resistance. Medicinal Research Reviews 2002, 22 (5) , 512-529.
    37. M. Maitrejean, G. Comte, J. M. Pérez-Victoria, F. Gamarro, A. Di Pietro, D. Barron. Flavonolignans from Silybum marianum L.. 2002, 329-335.
    38. Ahcène Boumendjel, Chantal Beney, Nabajyoti Deka, Anne-Marie Mariotte, Martin Ata Lawson, Doriane Trompier, Hélène Baubichon-Cortay, Attilio Di Pietro. 4-Hydroxy-6-methoxyaurones with High-Affinity Binding to Cytosolic Domain of P-Glycoprotein.. Chemical and Pharmaceutical Bulletin 2002, 50 (6) , 854-856.
    39. Gilles Comte, Jean-Baptiste Daskiewicz, Christine Bayet, Gwenaëlle Conseil, Armelle Viornery-Vanier, Charles Dumontet, Attilio Di Pietro, Denis Barron. C -Isoprenylation of Flavonoids Enhances Binding Affinity toward P-Glycoprotein and Modulation of Cancer Cell Chemoresistance. Journal of Medicinal Chemistry 2001, 44 (5) , 763-768.
    40. José M. Pérez-Victoria, F. Javier Pérez-Victoria, Gwenaëlle Conseil, Mathias Maitrejean, Gilles Comte, Denis Barron, Attilio Di Pietro, Santiago Castanys, Francisco Gamarro. High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin. Antimicrobial Agents and Chemotherapy 2001, 45 (2) , 439-446.
    41. Mathias Maitrejean, Gilles Comte, Denis Barron, Karim El Kirat, Gwenaëlle Conseil, Attilio Di Pietro. The flavanolignan silybin and its hemisynthetic derivatives, a novel series of potential modulators of p-glycoprotein. Bioorganic & Medicinal Chemistry Letters 2000, 10 (2) , 157-160.
    42. Jacques Ferté. Analysis of the tangled relationships between P‐glycoprotein‐mediated multidrug resistance and the lipid phase of the cell membrane. European Journal of Biochemistry 2000, 267 (2) , 277-294.
    43. Frances J. Sharom, Ronghua Liu, Yolanda Romsicki, Peihua Lu. Insights into the structure and substrate interactions of the P-glycoprotein multidrug transporter from spectroscopic studies. Biochimica et Biophysica Acta (BBA) - Biomembranes 1999, 1461 (2) , 327-345.
    44. Frédéric Bois, Ahcène Boumendjel, Anne-Marie Mariotte, Gwenaëlle Conseil, Attilio Di Petro. Synthesis and biological activity of 4-alkoxy chalcones: potential hydrophobic modulators of p-glycoprotein-mediated multidrug resistance. Bioorganic & Medicinal Chemistry 1999, 7 (12) , 2691-2695.
    45. José M. Pérez-Victoria, Benigna M. Tincusi, Ignacio A. Jiménez, Isabel L. Bazzocchi, Mahabir P. Gupta, Santiago Castanys, Francisco Gamarro, Angel G. Ravelo. New Natural Sesquiterpenes as Modulators of Daunomycin Resistance in a Multidrug-Resistant Leishmania t ropica Line ,. Journal of Medicinal Chemistry 1999, 42 (21) , 4388-4393.
    46. A. Di Pietro, G. Dayan, G. Conseil, E. Steinfels, T. Krell, D. Trompier, H. Baubichon-Cortay, J.-M. Jault. P-glycoprotein-mediated resistance to chemotherapy in cancer cells: using recombinant cytosolic domains to establish structure-function relationships. Brazilian Journal of Medical and Biological Research 1999, 32 (8) , 925-939.
    47. Frédéric Bois, Chantal Beney, Ahcène Boumendjel, Anne-Marie Mariotte, Gwenaëlle Conseil, Attilio Di Pietro. Halogenated Chalcones with High-Affinity Binding to P-Glycoprotein:  Potential Modulators of Multidrug Resistance. Journal of Medicinal Chemistry 1998, 41 (21) , 4161-4164.
    48. Gwenaelle Conseil, Helene Baubichon-Cortay, Guila Dayan, Jean-Michel Jault, Denis Barron, Attilio Di Pietro. Flavonoids: A class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein. Proceedings of the National Academy of Sciences 1998, 95 (17) , 9831-9836.
    49. Véronique Marsaud, Christine Mercier-Bodard, Dominique Fortin, Stéphane Le Bihan, Jack-Michel Renoir. Dexamethasone and triamcinolone acetonide accumulation in mouse fibroblasts is differently modulated by the immunosuppressants cyclosporin A, FK506, rapamycin and their analogues, as well as by other P-glycoprotein ligands. The Journal of Steroid Biochemistry and Molecular Biology 1998, 66 (1-2) , 11-25.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    Pair your accounts.

    Export articles to Mendeley

    Get article recommendations from ACS based on references in your Mendeley library.

    You’ve supercharged your research process with ACS and Mendeley!

    STEP 1:
    Click to create an ACS ID

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Please note: If you switch to a different device, you may be asked to login again with only your ACS ID.

    Your Mendeley pairing has expired. Please reconnect