Development and application of novel drugs frequently fail due to adverse changes of TH-levels or thyroid histology in animal experiments. Such effects potentially arise from interference with thyroid gland function or key regulators of local thyroid hormone (TH) transport, metabolism and action. Deiodinases are key enzymes in TH in-/activation, with impact on development, cell differentiation and energy metabolism among other physiological processes, and have therefore been identified as molecular initiating events (MIE) in the assessment of chemicals for endocrine disruption.
A robust semi-automatic 384-well High Throughput Screening (HTS) platform was developed and now employed for screening substances interfering with DIO2 activity, utilizing the non-radioactive Sandell-Kolthoff (SK) reaction to determine DIO-depending iodide release. In an initial HTS using recombinantly expressed human DIO2, 1759 components from a FDA-approved drug library were tested at a single concentration of 20 µM.
At the given concentration, 1.1% of the tested drugs showed DIO2 inhibition by >25% and 0.2 % an inhibition by > 50%. 11.5% of the screened drugs had characteristics of either potential DIO2 activators or SK interference. Among the inactive compounds were some well-known DIO1-selective inhibitors such as PTU or Genistein, a finding which supports the specific and predictive quality of this HTS approach.
Conclusion & outlook
In this pilot study, we were able to demonstrate that even a library of FDA-approved drugs does contain compounds that may exert adverse effects on TH metabolism by DIO2 inhibition. In perspective, such compound should be revised regarding potential side effects that might appear from such interference, e.g. abnormal patterning of TH metabolites in serum or defects that might appear from local suppression of DIO2 activity. While this screening setup is appropriate to detect endocrine disruptors (ED) affecting DIO2 activity, it does, on the other hand, also opens the perspective to identify highly potent drug candidates and reference compounds for ED research. In summary, this study represents a major success in the development of an in vitro strategy to identify endocrine disruptors, characterized by inhibition of deiodinases. The established 384-well assay protocol also provides the basis for further testing of large chemical libraries against DIO2, and can be adapted to other deiodinating enzymes modulating TH availability, e.g. DIO1 or dehalogenase (IYD). Furthermore, generated data pools provide the basis to select Quantitative Structure Activity Relationship training sets to develop predictive in silico tools for preselection and toxicological assessment.
Supported by ATHENA EU-Grant nº825161.
22 May 2021 - 26 May 2021