Estébanez-Perpiñá Eva, Arnold Leggy A, Jouravel Natalia, Togashi Marie, Blethrow Justin, Mar Ellena, Nguyen Phuong, Phillips Kevin J, Baxter John D, Webb Paul, Guy R Kiplin, Fletterick Robert J
Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158-2240, USA.
Mol Endocrinol. 2007 Dec;21(12):2919-28. doi: 10.1210/me.2007-0174. Epub 2007 Sep 6.
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.
直接抑制核激素受体与其必需共激活因子结合的核激素受体拮抗剂的开发，将有助于对核激素受体信号传导进行有效调控。我们之前在一次高通量筛选中鉴定出3-（二丁基氨基）-1-（4-己基苯基）-丙-1-酮（DHPPA），一种芳香族β-氨基酮，它能抑制共激活因子与甲状腺激素受体β（TRβ）的结合。初步证据表明，能使TRβ表面特定半胱氨酸残基烷基化的芳香族β-烯酮1-（4-己基苯基）-丙-2-烯-1-酮（HPPE）是从DHPPA中释放出来的。然而，DHPPA作用机制和特异性的一些方面仍不清楚。在此，我们报道了TRβ与抑制剂HPPE在2.3埃分辨率下的X射线晶体结构。未反应的HPPE位于通常介导TRβ与其共激活因子结合的界面处。包括TRβ突变体实验和质谱分析在内的多项证据表明，HPPE特异性地使位于激活功能-2口袋附近的TRβ的半胱氨酸残基298烷基化。我们提出这种共价加合物的形成通过两步机制进行：1）β-消除形成HPPE；2）HPPE与TRβ之间缓慢形成共价键。DHPPA代表了一类新型的强效TRβ拮抗剂，其晶体结构为设计靶向核激素受体基因调控复合物组装并阻断转录的拮抗剂提供了新方法。
