Novel Deiodinase 2-Selective Inhibitors-Possible Reference Substances for Regulatory In Vitro Tests for Endocrine...

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. Three selenoprotein isoenzymes, deiodinases 1-3 (DIO1-3), are essential components of TH metabolism, and their activities have been identified as relevant endpoints regarding the screening of compounds influencing the TH system. Given the importance of DIO2 as the key enzyme for local activation of the prohormone T4 to the active T3 in various tissues, and limited data on selective biochemical DIO2 inhibition, there is a clear need to identify potent and selective DIO2-inhibiting compounds.

Human-recombinant DIO2 enzyme pools were prepared from HEK293 cells overexpressing DIO2 and used as a robust enzyme source for the development, optimization, and semiautomated miniaturization of a nonradioactive DIO2 high-throughput screening (HTS) enzyme assay for the identification of DIO2-selective small molecule inhibitors. LT4 was used as substrate, and enzymatic release of iodide was colorimetrically quantified by the iodide-catalyzed Sandell-Kolthoff reaction. Eight comprehensive small molecule libraries were screened, covering ∼1/5 of the synthetic chemicals currently registered, natural products, as well as FDA-approved drugs. A total of 59,928 compounds were first screened at a single 10 µM concentration, followed by a validation screen to confirm the primary hits. Subsequently, DIO isoenzyme selectivity and cytotoxicity were evaluated.

Utilizing this highly reproducible and robust HTS test system with a determined median Z'-factor of 0.70 identified 356 primary inhibitory hits. Concentration-response experiments verified 17 potent inhibitors, further characterized regarding their DIO isoenzyme selectivity and cytotoxicity. Six potent DIO2-selective inhibitors, including two FDA-approved drugs and various novel pan-DIO inhibitors, for example, the fungicide fluazinam, were identified.

Specific DIO2 inhibitors, such as the FDA-approved drugs racecadotril and ibrutinib and the tyrosine kinase inhibitor rociletinib, might serve as a future toolbox for reversible pharmacological interference with the local provision of DIO2-generated T3 from T4 during development, tissue regeneration, and various DIO2-dependent metabolic processes. Furthermore, they can serve as reference compounds for the development and validation of regulatory in vitro tests. Identified FDA-approved drugs warrant a closer look at potential disturbances of local TH availability.