Discovery of diverse thyroid hormone receptor antagonists by high-throughput docking

Abstract

Treatment of hyperthyroidism, a common clinical condition that can have serious manifestations in the elderly, has remained essentially unchanged for >30 years. Directly antagonizing the effect of the thyroid hormone at the receptor level may be a significant improvement for the treatment of hyperthyroid patients. We built a computer model of the thyroid hormone receptor (TR) ligand-binding domain in its predicted antagonist-bound conformation and used a virtual screening algorithm to select 100 TR antagonist candidates out of a library of >250,000 compounds. We were able to obtain 75 of the compounds selected in silico and studied their ability to act as antagonists by using cultured cells that express TR. Fourteen of these compounds were found to antagonize the effect of T3 on TR with IC50s ranging from 1.5 to 30 microM. A small virtual library of compounds, derived from the highest affinity antagonist (1-850) that could be rapidly synthesized, was generated. A second round of virtual screening identified new compounds with predicted increased antagonist activity. These second generation compounds were synthesized, and their ability to act as TR antagonists was confirmed by transfection and receptor binding experiments. The extreme structural diversity of the antagonist compounds shows how receptor-based virtual screening can identify diverse chemistries that comply with the structural rules of TR antagonism.

Fig. 3.

Chemical synthesis of 1-850 derivatives was conducted by preparing a nonvariable core structure and rapidly linking commercially available polysubstituted phenylisocyanate building blocks in a simple step of parallel synthesis (see Materials and Methods for details).

Fig. 1.

Inhibition of [¹²⁵I]T3 binding to TRs by 1-850 in intact cells. The GH4C1 pituitary cell line, which contains endogenous TRs (TRα, TRβ1, and TRβ2), was incubated with 0.1 nM [¹²⁵I]T3 alone and with the indicated concentrations of unlabeled T3 and 1-850 as described in Materials and Methods. After incubation for 60 min at 37°C, the cells were chilled and washed, and the nuclei were isolated as described in Materials and Methods. The results indicate the inhibition of binding of [¹²⁵I]T3 by T3 and 1-850.

Fig. 2.

Predicted conformation of 1-850 (gold) bound to the TRβ ligand-binding pocket. (Upper) A hydrogen bond between His-435 and a carbonyl oxygen of 1-850 and possibly between Arg-282 and a nitro oxygen of 1-850 constitute the only polar interactions. All other contacts are hydrophobic (not shown for clarity). (Lower)1-850 superimposes with the crystal structure of T3 (green), bound to active TR and clashes with the active conformation of helix H12 (cyan). Color code is red for oxygen, blue for nitrogen, and magenta for fluoride/iodide in 1-850/T3, respectively.

Fig. 4.

Comparison of the antagonist activity of 1-850 and two compounds (D1 and D4) identified through a VLS of derivatives of 1-850.

Fig. 5.

Inhibition of T3-mediated coactivator recruitment to TR by D1, D4, and 1-850 in vitro. Approximately 2.5–5 × 10⁴ cpm of ³⁵S-labeled TRα (20 fmol) in 2 μl of lysate was incubated with 500 ng of GST fused to the receptor-interaction region of the coactivator NRC (NRC15) immobilized on glutathione-agarose beads. The samples were also incubated for 15 min at room temperature with 2 μM D1 or D4 or 5 μM 1-850 in binding buffer as indicated in Materials and Methods. The samples were then chilled on ice and incubated with 1 nM T3 for an additional 60 min at 4°C. Control samples contained no T3 or antagonists or received only T3. The beads were washed and the bound [³⁵S]TRα was electrophoresed in a an SDS/10% polyacrylamide gel followed by analysis and quantitation of the amount of [³⁵S]TRα bound by using a Molecular Dynamics PhosphorImager and imagequant software. The percent inhibition of T3-mediated binding of [³⁵S]TRα to GST-NRC15 by 1-850, D1, and D4 was determined after subtracting the amount of [³⁵S]TRα bound to GST-NRC15 in the absence of T3.