Department of Oncology (C.S., S.K., Y.H., L.P., M.R.) and Smart Sensors and Integrated Microsystems (SSIM) Program (S.Y., G.A.), Wayne State University School of Medicine and Barbara Ann Karmanos Cancer Institute, Detroit, Michigan; Department of Biochemistry and Molecular Biology, College of Natural Science, Michigan State University, East Lansing, Michigan (N.I. and A.D.); School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom (C.D. and P.E.S.); and Department of Pharmacology, UNC-Chapel Hill School of Medicine, Chapel Hill, North Carolina (N.N.).
Department of Oncology (C.S., S.K., Y.H., L.P., M.R.) and Smart Sensors and Integrated Microsystems (SSIM) Program (S.Y., G.A.), Wayne State University School of Medicine and Barbara Ann Karmanos Cancer Institute, Detroit, Michigan; Department of Biochemistry and Molecular Biology, College of Natural Science, Michigan State University, East Lansing, Michigan (N.I. and A.D.); School of Life Sciences, University of Nottingham, Queens Medical Centre, Nottingham, United Kingdom (C.D. and P.E.S.); and Department of Pharmacology, UNC-Chapel Hill School of Medicine, Chapel Hill, North Carolina (N.N.)
Mol Pharmacol. 2023 Apr;103(4):211-220. doi: 10.1124/molpharm.122.000589. Epub 2023 Jan 31.
The androgen receptor (AR) is a crucial coactivator of ELK1 for prostate cancer (PCa) growth, associating with ELK1 through two peptide segments (358-457 and 514-557) within the amino-terminal domain (NTD) of AR. The small-molecule antagonist 5-hydroxy-2-(3-hydroxyphenyl)chromen-4-one (KCI807) binds to AR, blocking ELK1 binding and inhibiting PCa growth. We investigated the mode of interaction of KCI807 with AR using systematic mutagenesis coupled with ELK1 coactivation assays, testing polypeptide binding and Raman spectroscopy. In full-length AR, deletion of neither ELK1 binding segment affected sensitivity of residual ELK1 coactivation to KCI807. Although the NTD is sufficient for association of AR with ELK1, interaction of the isolated NTD with ELK1 was insensitive to KCI807. In contrast, coactivation of ELK1 by the AR-V7 splice variant, comprising the NTD and the DNA binding domain (DBD), was sensitive to KCI807. Deletions and point mutations within DBD segment 558-595, adjacent to the NTD, interfered with coactivation of ELK1, and residual ELK1 coactivation by the mutants was insensitive to KCI807. In a lutathione -ransferase pull-down assay, KCI807 inhibited ELK1 binding to an AR polypeptide that included the two ELK1 binding segments and the DBD but did not affect ELK1 binding to a similar AR segment that lacked the sequence downstream of residue 566. Raman spectroscopy detected KCI807-induced conformational change in the DBD. The data point to a putative KCI807 binding pocket within the crystal structure of the DBD and indicate that either mutations or binding of KCI807 at this site will induce conformational changes that disrupt ELK1 binding to the NTD. SIGNIFICANCE STATEMENT: The small-molecule antagonist KCI807 disrupts association of the androgen receptor (AR) with ELK1, serving as a prototype for the development of small molecules for a novel type of therapeutic intervention in drug-resistant prostate cancer. This study provides basic information needed for rational KCI807-based drug design by identifying a putative binding pocket in the DNA binding domain of AR through which KCI807 modulates the amino-terminal domain to inhibit ELK1 binding.
雄激素受体（AR）是前列腺癌（PCa）生长的 ELK1 的关键共激活因子，通过 AR 氨基端结构域（NTD）内的两个肽段（358-457 和 514-557）与 ELK1 结合。小分子拮抗剂 5-羟基-2-（3-羟基苯基）色满-4-酮（KCI807）与 AR 结合，阻止 ELK1 结合并抑制 PCa 生长。我们使用系统诱变与 ELK1 共激活测定相结合，研究了 KCI807 与 AR 的相互作用模式，测试了多肽结合和拉曼光谱。在全长 AR 中，缺失 ELK1 结合片段都不会影响残留的 ELK1 共激活对 KCI807 的敏感性。尽管 NTD 足以与 ELK1 结合，但分离的 NTD 与 ELK1 的相互作用对 KCI807 不敏感。相反，AR-V7 剪接变体（包含 NTD 和 DNA 结合域（DBD））的 ELK1 共激活对 KCI807 敏感。DBD 片段 558-595 内的缺失和点突变，紧邻 NTD，干扰 ELK1 的共激活，并且突变体的残留 ELK1 共激活对 KCI807 不敏感。在谷胱甘肽转移酶下拉测定中，KCI807 抑制了包含两个 ELK1 结合片段和 DBD 的 AR 多肽与 ELK1 的结合，但不影响与缺乏残基 566 下游序列的类似 AR 片段的 ELK1 结合。拉曼光谱检测到 KCI807 诱导 DBD 构象变化。数据指向 DBD 晶体结构中的推定 KCI807 结合口袋，并表明该位点的突变或 KCI807 的结合将诱导构象变化，从而破坏 ELK1 与 NTD 的结合。意义陈述：小分子拮抗剂 KCI807 破坏了雄激素受体（AR）与 ELK1 的结合，为开发用于耐药性前列腺癌的新型治疗干预的小分子提供了原型。这项研究通过鉴定 AR 的 DNA 结合域中的一个假定结合口袋，为基于 KCI807 的合理药物设计提供了基本信息，通过该口袋，KCI807 调节氨基端结构域以抑制 ELK1 结合。
