title: Phenytoin is an estrogen receptor α-selective modulator that interacts with helix 12.
abstract: BACKGROUND
  Phenytoin (Dilantin(®); DPH) is used to treat epilepsy but causes estrogen agonist-antagonist-like side effects. We investigated the interaction of phenytoin with estrogen receptors (ERs) α and β by computational molecular docking, ER competition binding, transcriptional assays, and biological actions, comparing outcomes with estradiol (E2), estrone (E1), and tamoxifen (TMX).
  
  
  METHODS
  (1) The DPH docking to 3-dimensional crystal structures of the ERα ligand-binding domain (LBD) showed a high degree of structural complementarity (-57.15 calculated energy units, approximating kcal/mol) with the ligand-binding pocket, including a contact at leucine (L540) in helix 12. Estrogen receptor β showed slightly less favorable interactions (-54.27 kcal/mol), without contacting L450. Estradiol, E1, and TMX contact points with ERα and ERβ do not include L450. (2) Cellular actions: Incubation of cells transfected with ERα or ERβ and a luciferase promoter phenytoin was several orders weaker than E2 as an agonist through ERα and had no effect through ERβ. However, phenytoin at clinical concentrations (10(-11) to 10(-6) mol/L) powerfully antagonized action of E2 on ERα-expressing cells. Similarly, phenytoin at clinically effective concentrations marginally induced alkaline phosphatase by ERα- and ERβ-expressing endometrial cancer cells but at doses well below clinical effectiveness blocked E2-induced alkaline phosphatase. (3) ER competition: In Scatchard plots comparing phenytoin with 17β-estradiol against endometrial cancer cell cytosol E2-alone more effectively displaced labeled E2 than phenytoin, but phenytoin was approximately equimolar effective to E2 in inhibiting E2's displacement of the radiolabel, further confirming that phenytoin is a strong E2 antagonist.
  
  
  CONCLUSIONS
  At clinically effective concentrations, phenytoin is a strong ERα cell antagonist but a many-fold weaker agonist. Although it interacts with ERβ LBD residues, phenytoin has no effects on ERβ-only expressing cells. Docking studies indicate phenytoin interacts with the ERα LBD at the hinge of helix 12 and could thereby interfere with the entry of other ER ligands or with the mobility of helix 12, either of which actions could explain phenytoin's antagonism of ER-mediated E2 actions. Our results suggest an explanation for the broad profile of phenytoin's actions and raise possibilities for the use of phenytoin or congeners in the clinical management of ERα-dependent conditions.
authors.affiliation: Department of Obstetrics and Gynecology, New York University School of Medicine, New York University, New York, NY, USA.
authors.forename: A
authors.initials: A
authors.lastname: Fadiel
authors.affiliation: Department of Obstetrics and Gynecology, New York University School of Medicine, New York University, New York, NY, USA.
authors.forename: J
authors.initials: J
authors.lastname: Song
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York University, New York, NY, USA.
authors.forename: D
authors.initials: D
authors.lastname: Tivon
authors.affiliation: School of Pharmacy, University of Kentucky, Lexington, KY, USA.
authors.forename: A
authors.initials: A
authors.lastname: Hamza
authors.affiliation: Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York University, New York, NY, USA.
authors.forename: T
authors.initials: T
authors.lastname: Cardozo
authors.affiliation: Department of Obstetrics and Gynecology, New York University School of Medicine, New York University, New York, NY, USA frederick.naftolin@nyumc.org.
authors.forename: Frederick
authors.initials: F
authors.lastname: Naftolin
mesh_terms: D000595:Amino Acid Sequence; D000927:Anticonvulsants / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D001667:Binding, Competitive; D003461:Crystallography; D004305:Dose-Response Relationship, Drug; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D047629:Estrogen Receptor beta / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004970:Estrone / Q000378:metabolism / Q000494:pharmacology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D061986:MCF-7 Cells; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D010672:Phenytoin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D020845:Selective Estrogen Receptor Modulators / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D013629:Tamoxifen / Q000378:metabolism / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D000927:Anticonvulsants; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D020845:Selective Estrogen Receptor Modulators; D013629:Tamoxifen; D004970:Estrone; D004958:Estradiol; D010672:Phenytoin
keywords: Dilantin®; diphenylhydantoin; epilepsy and computational molecular docking; estradiol; estrogen receptors