Thyroid hormone (TH) homeostasis depends on the coordination of several key events to maintain proper local TH signaling, including iodide uptake, hormone synthesis, metabolism, and elimination. The three isoenzymes, Deiodinases (DIO1-3), are essential components of hormone metabolism. They catalyze iodide release the outer and/or inner ring of TH structure to convert between their active and inactive forms. The activity of DIOs has been identified as an important endpoint regarding the screening of compounds for TH system disruption. The classical DIO assays rely on the liberation of radioactive iodide from a tracer substrate. Furthermore, substrate deiodination can also be followed by using mass spectrometry as readout. However, radioactively labeled substrate molecules are expensive, not always commercially available, legally restricted and limited in their half-life. Mass spectrometry involves costly and sensitive instrumentation and skilled personnel. To avoid these difficulties, we established a cost- and time-effective semi-automatic, non-radioactive 96-well DIO2 assay, based on the Sandell-Kolthoff (SK) reaction, and adapted it to the 384-well High Throughput Screening (HTS) plate format to identify inhibitors or activators of DIO2 activity from a small molecule compound library. A robust 384-well HTS platform for non-radioactive determination of DIO2-catalyzed iodide release by the SK method was developed. The SK reaction describes the reduction of yellow-colored cerium IV to non-colored cerium III by arsenic III. Reaction rate is increased in a concentration-dependent manner by presence of iodide and can be quantified photometrically. The development process included optimization of protein concentration using recombinantly expressed human DIO2 and incubation conditions (time, temperature, shaking, kinetics). Furthermore, assay performance parameters were examined such as linear measuring detection range and Z-factor. Several steps of the protocol were performed on and optimized for the Biomek Workstation (Beckman Coulter) platform. 50 µg total protein/reaction and 4 h incubation time at 37°C with an endpoint measurement at OD 20 min delivered the most robust parameters of the semi-automatic, non-radioactive 384-well HTS DIO2 assay with a Z-factor consistently above 0.5. The presented work demonstrated the successful setup of an HTS-screening platform, qualified to detect and initially characterize endocrine disruptors, affecting DIO activities. The respective HTS protocol provides the basis for further testing of large chemical libraries against DIO2, and can be adapted to other deiodinating enzymes, e.g. DIO1 and dehalogenases. Furthermore, its use to generate large data sets on libraries covering diverse chemical structures will enable the setup of predictive in silico tools by providing respective training sets.
Supported by ATHENA EU-Grant nº825161.
22 May 2021 - 26 May 2021