Latif Rauf, Realubit Ronald B, Karan Charles, Mezei Mihaly, Davies Terry F
Thyroid Research Unit, James J. Peters VA Medical Center, Icahn School of Medicine at Mount Sinai , New York, NY , USA.
Sulzberger Columbia Genome Center, Columbia University , New York, NY , USA.
Front Endocrinol (Lausanne). 2016 Sep 27;7:130. doi: 10.3389/fendo.2016.00130. eCollection 2016.
Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves' disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a score of 0.3-0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin - a post receptor activator of adenylyl cyclase - confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has the potential to be developed as a therapeutic antagonist for abrogation of TSHR signaling by TSHR autoantibodies in GD.
格雷夫斯病（GD）患者体内的促甲状腺激素受体（TSHR）的病理激活是由促甲状腺激素抗体引起的，或者是由体细胞和罕见的基因组突变导致的，这些突变增强了受体的组成性激活，影响G蛋白和非G蛋白信号传导。潜在的选择性小分子拮抗剂代表了用于消除此类异常TSHR信号传导的新型治疗化合物。在本研究中，我们描述了通过高通量筛选（HTS）鉴定和表征一种新型小分子拮抗剂的过程。TSHR拮抗剂的鉴定是使用基于转录的TSH抑制生物测定法进行的。表达TSHR的CHO细胞，其也表达了荧光素酶标记的CRE反应元件，以牛TSH作为激活剂，在384孔板中进行优化，其得分在0.3-0.6之间。使用这种HTS测定法，我们以16.7μM的终浓度筛选了约80,000种化合物的多样化文库。阳性命中的选择标准基于对照TSH刺激抑制≥50％的平均信号阈值。筛选产生了450个阳性命中，命中率为0.56％。针对TSH和福斯可林（一种腺苷酸环化酶的受体后激活剂）的二次确认筛选证实了一种TSHR特异性候选拮抗剂分子（命名为VA-K-14）。这种先导分子的IC为12.3μM，具有独特的化学结构。对细胞活力的平行分析表明，先导抑制剂在其有效浓度下无细胞毒性。使用TSHR跨膜模型进行的对接研究显示了疏水接触位置和TSHR信号传导的可能抑制模式。此外，该分子能够抑制GD患者血清和单克隆刺激TSHR抗体对TSHR的刺激。总之，我们报告了一种新型小分子TSHR抑制剂的鉴定，该抑制剂有可能被开发为一种治疗拮抗剂，用于消除GD中TSHR自身抗体引起的TSHR信号传导。
