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Probing the structural basis of P‐glycoprotein transport of μ‐opioid receptor agonists: methadone and loperamide
Author(s) -
Gibbs Morgan Elizabeth,
Ledwitch Kaitlyn,
Roberts Arthur
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.950.6
Subject(s) - loperamide , chemistry , pharmacology , p glycoprotein , agonist , methadone , opioid , transporter , efflux , receptor , biochemistry , medicine , diarrhea , multiple drug resistance , gene , antibiotics
P‐glycoprotein (Pgp) protects the brain from toxic insults by acting as a gatekeeper at the blood‐brain barrier. In addition to effluxing toxins, Pgp can also limit penetration of therapeutics into the brain for the treatment of central nervous system (CNS) disorders and diseases, including μ‐opioid receptor agonists that are used in pain management. Although they are structurally similar, the μ‐opioid receptor agonist loperamide (Imodium®) is effluxed out of the CNS by Pgp at four times the rate of methadone. Because of these differences, methadone and loperamide were used as model drugs to probe the mechanism of Pgp‐mediated μ‐opioid receptor agonist efflux. We hypothesize that higher Pgp‐mediated efflux rates of loperamide are the result of multiple loperamide binding to Pgp, allowing more drug to be transported per ATP hydrolyzed. We found that loperamide was a more potent activator of Pgp‐mediated ATP hydrolysis than methadone. Consistent with our hypothesis, the loperamide‐induced activation of ATP hydrolysis suggested at least two loperamide binding sites on Pgp, while methadone‐induced activation of ATP hydrolysis suggested a single methadone binding site. NMR and fluorescence were used to investigate the interactions of these drugs with the transporter and develop a structural model for transport. The saturation transfer double difference (STDD) NMR technique identified the functional groups involved in molecular recognition of Pgp and revealed that loperamide had more interactions with the transporter than methadone, which is consistent with the higher transport rate of loperamide. Acrylamide quenching of Pgp fluorescence to probe transporter conformation indicated that loperamide shifts Pgp into a conformation that promotes ATP hydrolysis and transport, while methadone shifts Pgp into an inhibitory conformation. This information combined with paramagnetic relaxation enhancement (PRE) NMR experiments and computer modelling was used to pinpoint the binding locations of loperamide and methadone on Pgp. The drug‐bound Pgp models suggest that multiple drug binding and conformational changes play a critical role in Pgp transport of this class of drugs. Support or Funding Information This work was funded and supported by the National Institute of Health R15 Area Grant (1R15GM107913‐01A1).