title: An Improved Nonradioactive Screening Method Identifies Genistein and Xanthohumol as Potent Inhibitors of Iodothyronine Deiodinases.
abstract: BACKGROUND
  Deiodinases (DIO1, 2, and 3) are key enzymes in thyroid hormone (TH) activation and inactivation with impact on energy metabolism, development, cell differentiation, and a number of other physiological processes. The three DIO isoenzymes thus constitute sensitive rate-limiting components within the TH axis, prone to dysregulation by endocrine disruptive compounds or disease state. In animal models and cell culture experiments, they serve as readout for local TH status and disarrangement of the hormonal axis. Furthermore, some human diseases are characterized by apparent deiodinase dysregulation (e.g., the low triiodothyronine syndrome in critical illness). Consequently, these enzymes are targets of interest for the development of pharmacological compounds with modulatory activities. Until now, the portfolio of inhibitors for these enzymes is limited. In the clinics, the DIO1-specific inhibitor propylthiouracil is in use for treatment of severe hyperthyroidism. Other well-known inhibitors (e.g., iopanoic acid or aurothioglucose) are nonselective and block all three isoenzymes. Furthermore, DIO3 was shown to be a potential oncogenic gene, which is strongly expressed in some tumors and might, in consequence, protect tumor tissue form differentiation by TH. With respect to its role in tumorigenesis, specific inhibitors of DIO3 as a potential target for anticancer drugs would be highly desirable. To this end, a flexible and convenient assay for high-throughput screening is needed. We recently described a nonradioactive screening assay, utilizing the classic Sandell-Kolthoff reaction as readout for iodide release from the substrate molecules. While we used murine liver as enzyme source, the assay was limited to murine DIO1 activity testing. Here, we describe the use of recombinant proteins as enzyme sources within the assay, expanding its suitability from murine Dio1 to human DIO1, DIO2, and DIO3.
  
  
  METHODS
  As proof-of-concept, deiodination reactions catalyzed by these recombinant enzymes were monitored with various nonradioactive substrates and confirmed by liquid chromatography-tandem mass spectrometry.
  
  
  RESULTS
  The contrast agent and known DIO inhibitor iopanoic acid was characterized as readily accepted substrate by DIO2 and Dio3. In a screening approach using established endocrine disrupting compounds, the natural food ingredient genistein was identified as a further DIO1-specific inhibitor, while xanthohumol turned out to potently block the activity of all three isoenzymes.
  
  
  CONCLUSIONS
  A rapid nonradioactive screening method based on the Sandell-Kolthoff reaction is suitable for identification of environmental, nutritive and pharmacological compounds modulating activities of human deiodinase enzymes.
authors.affiliation: 1 Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Kostja
authors.initials: K
authors.lastname: Renko
authors.affiliation: 1 Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Sonja
authors.initials: S
authors.lastname: Schäche
authors.affiliation: 2 Karolinska Institutet , Department of Cellular and Molecular Biology, Stockholm, Sweden .
authors.forename: Carolin S
authors.initials: CS
authors.lastname: Hoefig
authors.affiliation: 1 Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Tim
authors.initials: T
authors.lastname: Welsink
authors.affiliation: 1 Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Christian
authors.initials: C
authors.lastname: Schwiebert
authors.affiliation: 3 Institut für Biochemie und Molekularbiologie, Rheinische Friedrich Wilhelms-Universität Bonn, Bonn , Germany .
authors.forename: Doreen
authors.initials: D
authors.lastname: Braun
authors.affiliation: 1 Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Niels-Peter
authors.initials: NP
authors.lastname: Becker
authors.affiliation: 1 Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Josef
authors.initials: J
authors.lastname: Köhrle
authors.affiliation: 1 Institut für Experimentelle Endokrinologie, Charité - Universitätsmedizin Berlin , Berlin, Germany .
authors.forename: Lutz
authors.initials: L
authors.lastname: Schomburg
mesh_terms: D000818:Animals; D002384:Catalysis; D002454:Cell Differentiation; D002853:Chromatography, Liquid; D004268:DNA-Binding Proteins / Q000737:chemistry; D004353:Drug Evaluation, Preclinical; D004798:Enzymes / Q000737:chemistry; D005419:Flavonoids / Q000627:therapeutic use*; D019833:Genistein / Q000627:therapeutic use*; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007453:Iodide Peroxidase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D007480:Iopanoic Acid / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry; D013058:Mass Spectrometry; D051379:Mice; D016366:Open Reading Frames; D011427:Propiophenones / Q000627:therapeutic use*; D011994:Recombinant Proteins / Q000737:chemistry; D013963:Thyroid Hormones / Q000737:chemistry; D000097564:Iodothyronine Deiodinase Type II
chemical_list: D004268:DNA-Binding Proteins; D004798:Enzymes; D005419:Flavonoids; D019833:Genistein; D007453:Iodide Peroxidase; D007480:Iopanoic Acid; D007527:Isoenzymes; D011427:Propiophenones; D011994:Recombinant Proteins; D013963:Thyroid Hormones; D000097564:Iodothyronine Deiodinase Type II; C518584:DIDO1 protein, human; C117274:iodothyronine deiodinase type III; C507436:selenodeiodinase type 1, mouse; C104536:xanthohumol