Screening assays on inhibitors, modulators and substrates of deiodinase and dehalogenase activities

Thyroid hormones (TH) are main players in regulation of development, differentiation and metabolism. Their biological activity is controlled by systemic availability and by local activation and inactivation reactions tightly controlling their temporal and spatial cell type-specific effects. The respective enzymes, i.e. the selenoprotein family of iodothyronine deiodinases (DIO), are thus potential targets for pharmacological interventions. Deiodination also occurs, when the precursor molecules of thyroid hormone synthesis, mono- and diiodotyrosine (MIT & DIT), are recycled within the thyroid. The responsible enzymes are the dehalogenases/iodotyronine deiodinases (IYD) and malfunction of this system leads to excessive iodine loss and goitrogenesis [1]. Direct in vitro monitoring of these activities classically utilizes a radioactive iodide-release assay with severe disadvantages regarding handling, time consumption, throughput, equipment and costs of waste management.

To overcome these limitations, we developed a non-radioactive screening variant of this assay suitable for microtiter format [2]. The new test depends on photometric measurement and follows the principle of the Sandell-Kolthoff-reaction. Recently, we have expanded the range of applicable activities to human DIO1, DIO2 and DIO3 by utilizing recombinant enzyme sources and adapted reaction conditions for recombinant IYD activity. Deiodinases were expressed in HEK293-cells and total protein was incubated under appropriate reaction conditions [2] with various iodothyronines as potential substrates. The deiodination pattern seen in the iodide-release assay were matched and verified by liquid chromatography-tandem mass spectrometry. Both methods showed the expected substrate preferences. Classical inhibitors were applied as negative controls (propylthiouracil for DIO1, aurothioglucose for DIO2 and DIO3). Monitoring of recombinant Dehal1 activity was possible under respective reaction conditions (modified from [3]). MIT and DIT were used as substrates and activity was inhibited by dibromotyrosine (DBT) and titratable with NADPH concentration. In conclusion, this photometric non-radioactive procedure allows rapid and sensitive screening of nutritional, environmental or pharmacological agents interfering in thyroid hormone metabolism in a microtiter plate assay format.

The study was supported by the Bundesministerium für Bildung und Forschung (BMBF # 0315370C).

[1] Grasberger, H., and Refetoff, S. (2011). Genetic causes of congenital hypothyroidism due to dyshormonogenesis. Current Opinion in Pediatrics August 2011 23, 421 – 428.

[2] Renko, K., Hoefig, C.S., Hiller, F., Schomburg, L., and Köhrle, J. (2012). Identification of iopanoic acid as substrate of type 1 deiodinase by a novel nonradioactive iodide-release assay. Endocrinology 153, 2506 – 2513.

[3] Gnidehou, S., Caillou, B., Talbot, M., Ohayon, R., Kaniewski, J., Noël-Hudson, M.-S., Morand, S., Agnangji, D., Sezan, A., Courtin, F., et al. (2004). Iodotyrosine dehalogenase 1 (DEHAL1) is a transmembrane protein involved in the recycling of iodide close to the thyroglobulin iodination site. FASEB J. 18, 1574 – 1576.