One of the many problems of hyperthyroidism is the limited number of medicines which act as anti-thyroid agents. The two groups that exist both have the potential for severe side effects/adverse reactions.
Any new agents have the potential for improving hyperthyroidism treatment. This is most especially true given that the accepted alternatives to the existing agents are radioactive iodine and thyroidectomy. Both of which are known to have the potential for numerous side effects and unintended consequences (e.g. damage to parathyroid glands, affecting the voice, salivary glands, etc.)
Even if these agents are not regarded as definitive, they might well allow safe long-term use.
Identification and Characterization of Highly Potent and Isoenzyme-Selective Inhibitors of Deiodinase Type I via a Nonradioactive High-Throughput Screening Method
Rajas Sane 1 , Carola Seyffarth 2 , Sabrina Kleissle 3 , Martin Neuenschwander 2 , Jens Peter von Kries 2 , Caroline Frädrich 1 , Kostja Renko 4 , Eva K Wirth 5 6 , Josef Köhrle 1
PMID: 40170637 DOI: 10.1089/thy.2025.0036
Abstract
Objective:
Deiodinase type I (DIO1) is crucial in maintaining thyroid hormone (TH) balance. It converts the prohormone thyroxine (T4) to the active triiodothyronine (T3) and degrades T3 to inactive 3,3'-diiodothyronine (3,3'-T2). It also acts on reverse T3 (rT3) and sulfated TH metabolites, thus contributing to TH elimination. Upregulation of DIO1 is linked to hyperthyroid conditions such as Graves' disease and autonomous thyroid adenoma, making it a promising target for pharmacological intervention. The adverse side effects of the antithyroid drug propylthiouracil (PTU), used in clinics to treat hyperthyroidism due to its thyroid peroxidase- and DIO1-blocking action, highlight the need for novel and potent DIO1-selective inhibitors.
Methods:
Using a semiautomatic high-throughput screening (HTS) assay based on the Sandell-Kolthoff (SK) reaction in 384-well plates, we screened 69,344 low-molecular-weight compounds for DIO1-inhibitory effects. Shortlisted hits underwent detailed manual characterization, where we evaluated the potency and isoenzyme specificity by assessing their DIO-inhibitory effects on enzyme preparations from all three DIO isoenzymes, over a wide concentration range (5 nM-20 µM). To evaluate the DIO1 inhibitory effects in intact cells, we applied a novel protocol based on the SK reaction to cell culture supernatants and assessed the intracellular deiodinase activity in DIO1 overexpressing HEK293 cells.
Results:
The robust HTS assay flagged 436 (<1%) of the screened compounds as hits, also including known DIO1 inhibitors such as PTU and genistein. Based on a validation screen of 298 compounds, we prioritized 26 compounds to comprehensively characterize their DIO1-selective inhibition. We identified 15 DIO1-selective compounds (IC50 < 1 µM), more potent than the bonafide DIO1-selective inhibitor PTU. Additionally, 8 of the 13 tested compounds were found capable of inhibiting DIO1 in intact cells.
Conclusions:
With a successful SK-reaction-based HTS application, we identified novel, potent, and selective inhibitors of DIO1 with nanomolar IC50 values. Furthermore, we successfully showed that some of these compounds were also capable of inhibiting intracellular DIO1 in intact cells. These novel compounds hold immense potential in studying TH modulation, deciphering DIO1 enzyme structure, and developing structure-activity relationships. Furthermore, our novel inhibitors act as lead compounds in developing strategies to combat hyperthyroidism.
Keywords: DIO1; DIO1-inhibitors; deiodinases; high-throughput screening; thyroid hormones.
