Structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic beta-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor beta (TR beta), in a high-throughput screen. Initial evidence suggested that the aromatic beta-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TR beta surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TR beta with the inhibitor HPPE at 2.3-angstrom resolution. Unreacted HPPE is located at the interface that normally mediates binding between TR beta and its coactivator. Several lines of evidence, including experiments with TR beta mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TR beta, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) beta-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TR beta. DHPPA represents a novel class of potent TR beta antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.