Discovery and Preclinical Characterization of NE-2-6 as a Potent Thyroid Peroxidase Inhibitor with Antithyroid Activity

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Graves’ disease is an autoimmune hyperthyroid disorder in which thyroid peroxidase (TPO) plays a central role in excessive thyroid hormone production. However, current TPO-targeting drugs, such as propylthiouracil, have limited selectivity and can cause serious adverse effects. In this study, we established an integrated discovery pipeline to identify and optimize novel small-molecule TPO inhibitors. The pipeline began with high-throughput screening of approximately 7000 compounds from the KRICT chemical library for peroxidase inhibition, followed by cytotoxicity filtering and iterative medicinal chemistry to generate NE-2 derivatives. Lead compounds (NE-2-6, NE-2-7, and NE-2-8) were evaluated using enzymatic assays, selectivity profiling against myeloperoxidase (MPO) and lactoperoxidase (LPO), molecular docking, plasma pharmacokinetic profiling, and in vivo antithyroid pharmacodynamic testing in an Ad-TSHR289-induced thyroid hyperfunction model. NE-2-6 exhibited potent TPO inhibitory activity and relative selectivity within the tested peroxidase panel. UV–visible spectral scanning and H2O2-dependent inhibition assays suggested that NE-2-6 perturbs the heme-associated catalytic environment of TPO; however, these data do not establish a definitive binding mode or kinetic inhibition mechanism. In the Ad-TSHR289-induced thyroid hyperfunction model, NE-2-6 reduced serum T4 levels, supporting antithyroid pharmacodynamic activity. Because autoimmune endpoints and dose-matched PK–PD relationships were not fully assessed, the findings should be interpreted as preliminary preclinical evidence supporting further evaluation of NE-2-6 as a TPO-targeting antithyroid candidate.