Mukherjee A, Kirkovsky L I, Kimura Y, Marvel M M, Miller D D, Dalton J T
Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee, Memphis 38163, USA.
Biochem Pharmacol. 1999 Oct 15;58(8):1259-67. doi: 10.1016/s0006-2952(99)00218-x.
We synthesized a series of potential chemoaffinity ligands for the androgen receptor (AR) by means of structural modifications of bicalutamide, a known nonsteroidal antiandrogen used in the treatment of hormone-dependent prostate cancer. We determined AR binding affinities of these ligands, identified chemoaffinity ligands by exchange assays, and confirmed irreversible binding to the AR by Scatchard analyses. AR binding affinity was determined in a competitive binding assay with a radiolabeled high-affinity AR ligand, [3H]mibolerone ([3H]MIB). For exchange assays, AR were incubated with an excess of each ligand, and then adsorbed onto hydroxyapatite (HAP). HAP-bound AR then were incubated with [3H]MIB to determine the remaining exchangeable specific binding sites. To determine the concentration of binding sites (Bmax), using Scatchard analysis, AR were incubated with a fixed concentration of ligand and increasing [3H]MIB concentrations. The ligands showed a wide range of AR binding affinities. In the exchange assays, three isothiocyanate derivatives of R-bicalutamide, the p-isothiocyanate (R-4), the p-thio-isothiocyanate (R-6), and the m-isothiocyanate (R-3), reduced exchangeable specific binding of [3H]MIB by 85, 84, and 50%, respectively. The S-isomer of p-thio-isothiocyanate (S-6), which showed 700-fold lower AR binding affinity than R-6, did not reduce exchangeable specific binding of [3H]MIB. In Scatchard analyses, the isothiocyanate derivatives R-3, R-4, and R-6 showed significant and progressive reduction in Bmax at increasing concentrations. The results indicate that initial specific reversible AR binding was required for subsequent covalent labeling, and that R-3, R-4, and R-6 bound the AR specifically and irreversibly. These isothiocyanate derivatives of R-bicalutamide are the first specific chemoaffinity ligands for the AR, and will provide valuable tools for the molecular characterization of the ligand binding domain of the AR.
我们通过对比卡鲁胺进行结构修饰，合成了一系列潜在的雄激素受体（AR）化学亲和配体。比卡鲁胺是一种已知的非甾体抗雄激素药物，用于治疗激素依赖性前列腺癌。我们测定了这些配体与AR的结合亲和力，通过交换试验鉴定化学亲和配体，并通过Scatchard分析确认其与AR的不可逆结合。在与放射性标记的高亲和力AR配体[3H]米勃龙（[3H]MIB）的竞争性结合试验中测定AR结合亲和力。对于交换试验，将AR与过量的每种配体孵育，然后吸附到羟基磷灰石（HAP）上。然后将结合在HAP上的AR与[3H]MIB孵育，以确定剩余的可交换特异性结合位点。为了确定结合位点的浓度（Bmax），使用Scatchard分析，将AR与固定浓度的配体和不断增加的[3H]MIB浓度孵育。这些配体显示出广泛的AR结合亲和力范围。在交换试验中，R-比卡鲁胺的三种异硫氰酸酯衍生物，即对异硫氰酸酯（R-4）、对硫代异硫氰酸酯（R-6）和间异硫氰酸酯（R-3），分别使[3H]MIB的可交换特异性结合降低了85%、84%和50%。对硫代异硫氰酸酯的S-异构体（S-6）与AR的结合亲和力比R-6低700倍，并未降低[3H]MIB的可交换特异性结合。在Scatchard分析中，异硫氰酸酯衍生物R-3、R-4和R-6在浓度增加时显示出Bmax的显著且逐渐降低。结果表明，后续的共价标记需要最初的特异性可逆AR结合，并且R-3、R-4和R-6特异性且不可逆地结合AR。这些R-比卡鲁胺的异硫氰酸酯衍生物是首批针对AR的特异性化学亲和配体，将为AR配体结合域的分子表征提供有价值的工具。
