Endocrine Abstracts

Objectives: Deiodinase type 1 (DIO1), a selenocysteine-containing enzyme belonging to the deiodinase (DIO) family, plays an essential role in the systemic and local modulation of thyroid hormone (TH) availability. However, DIO1 plays a dual role by not only converting the prohormone thyroxine (T4) to the active triiodothyronine (T3) but also degrading T3 to the inactive 3,3’-diiodothyronine (3,3’-T2). DIO1, by acting on sulfated TH metabolites, further contributes to TH inactivation and elimination. In the liver, T3 concentrations affect glucose, lipid, and cholesterol metabolism. Alterations to the local TH concentrations play a role in the progression of hepatic diseases; e.g., subclinical hypothyroidism is associated with the development of non-alcoholic fatty liver, and a hepatic TH drop might aggravate this disease. DIO1, the prominent hepatic deiodinase, is a critical target to modulate hepatic T3 concentrations. Therefore, by pharmacologically inhibiting hepatic DIO1 and its role as a T3-degrading enzyme, we hope to steer the local T3 concentrations and aid in the prevention and clearance of hepatic steatosis.

Methods: We used a High Throughput Screening (HTS) assay, based on iodide-catalysed Sandell-Kolthoff reaction, to identify novel and potent DIO1-selective inhibitors by screening 69344 small molecular weight compounds and using an enzyme preparation of human recombinant DIO1 (hrDIO1). We determined the potency and isoenzyme specificity of the shortlisted hits by testing them using enzyme preparations from all three human DIO isoenzymes over a wide inhibitor concentration range (5 nM - 20 µM). We further assessed these shortlisted candidates for their cytotoxic and DIO1-inhibitory effects on intact hrDIO1 overexpressing HEK293 cells.

Results: The HTS assay flagged known DIO1 inhibitors like propylthiouracil (PTU) and genistein, corroborating its efficacy. Based on a revalidation screen of 352 compounds, we prioritised 26 compounds to characterise the DIO1-selective inhibition comprehensively. We identified 15 DIO1-selective compounds (IC50s < 1 µM), that are more potent than the bonafide DIO1-selective inhibitor PTU, which also blocks thyroperoxidase. The shortlisted candidates exhibit either no or mild cytotoxic effects when incubated at 10 µM for 24 hours, and most are capable of inhibiting DIO1 in intact cells.

Conclusions: We successfully identified novel and highly potent DIO1-selective inhibitors whose IC50 values are in the nanomolar range. We characterised these inhibitors for their cytotoxicity and DIO1-inhibition in intact cells. Unravelling the role of hepatic T3 in lipid and energy metabolism and modulating T3 concentrations via DIO1 inhibitors may provide novel avenues for therapeutic intervention and exerting antisteatotic effects.