title: Structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor.
abstract: 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 (TRbeta), 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 TRbeta 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 TRbeta with the inhibitor HPPE at 2.3-A resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRbeta and its coactivator. Several lines of evidence, including experiments with TRbeta mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRbeta, 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 TRbeta. DHPPA represents a novel class of potent TRbeta 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158-2240, USA.
authors.forename: Eva
authors.initials: E
authors.lastname: Estébanez-Perpiñá
authors.forename: Leggy A
authors.initials: LA
authors.lastname: Arnold
authors.forename: Natalia
authors.initials: N
authors.lastname: Jouravel
authors.forename: Marie
authors.initials: M
authors.lastname: Togashi
authors.forename: Justin
authors.initials: J
authors.lastname: Blethrow
authors.forename: Ellena
authors.initials: E
authors.lastname: Mar
authors.forename: Phuong
authors.initials: P
authors.lastname: Nguyen
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Phillips
authors.forename: John D
authors.initials: JD
authors.lastname: Baxter
authors.forename: Paul
authors.initials: P
authors.lastname: Webb
authors.forename: R Kiplin
authors.initials: RK
authors.lastname: Guy
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fletterick
mesh_terms: D000096:Acetone / Q000737:chemistry / Q000494:pharmacology / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007659:Ketones / Q000737:chemistry; D008024:Ligands; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011988:Receptors, Thyroid Hormone / Q000037:antagonists & inhibitors* / Q000378:metabolism*