Data_Sheet_1_A Gq Biased Small Molecule Active at the TSH Receptor.docx

G protein coupled receptors (GPCRs) can lead to G protein and non-G protein initiated signals. By virtue of its structural property, the TSH receptor (TSHR) has a unique ability to engage different G proteins making it highly amenable to selective signaling. In this study, we describe the identification and characterization of a novel small molecule agonist to the TSHR which induces primary engagement with Gαq/11. To identify allosteric modulators inducing selective signaling of the TSHR we used a transcriptional-based luciferase assay system with CHO-TSHR cells stably expressing response elements (CRE, NFAT, SRF, or SRE) that were capable of measuring signals emanating from the coupling of Gαs, Gαq/11, Gβγ, and Gα12/13, respectively. Using this system, TSH activated Gαs, Gαq/11, and Gα12/13 but not Gβγ. On screening a library of 50K molecules at 0.1,1.0 and 10 μM, we identified a novel Gq/11 agonist (named MSq1) which activated Gq/11 mediated NFAT-luciferase >4 fold above baseline and had an EC50= 8.3 × 10−9 M with only minor induction of Gαs and cAMP. Furthermore, MSq1 is chemically and structurally distinct from any of the previously reported TSHR agonist molecules. Docking studies using a TSHR transmembrane domain (TMD) model indicated that MSq1 had contact points on helices H1, H2, H3, and H7 in the hydrophobic pocket of the TMD and also with the extracellular loops. On co-treatment with TSH, MSq1 suppressed TSH-induced proliferation of thyrocytes in a dose-dependent manner but lacked the intrinsic ability to influence basal thyrocyte proliferation. This unexpected inhibitory property of MSq1 could be blocked in the presence of a PKC inhibitor resulting in derepressing TSH induced protein kinase A (PKA) signals and resulting in the induction of proliferation. Thus, the inhibitory effect of MSq1 on proliferation resided in its capacity to overtly activate protein kinase C (PKC) which in turn suppressed the proliferative signal induced by activation of the predomiant cAMP-PKA pathway of the TSHR. Treatment of rat thyroid cells (FRTL5) with MSq1 did not show any upregulation of gene expression of the key thyroid specific markers such as thyroglobulin(Tg), thyroid peroxidase (Tpo), sodium iodide symporter (Nis), and the TSH receptor (Tshr) further suggesting lack of involvement of MSq1 and Gαq/11 activation with cellular differentation. In summary, we identified and characterized a novel Gαq/11 agonist molecule acting at the TSHR and which showed a marked anti-proliferative ability. Hence, Gq biased activation of the TSHR is capable of ameliorating the proliferative signals from its orthosteric ligand and may offer a therapeutic option for thyroid growth modulation.

G蛋白偶联受体（G protein coupled receptors, GPCRs）可启动G蛋白依赖及非G蛋白依赖的信号通路。促甲状腺激素受体（TSH receptor, TSHR）凭借其结构特性，能够结合多种不同的G蛋白，因此具备实现选择性信号转导的独特潜力。本研究中，我们对一种新型促甲状腺激素受体小分子激动剂进行了鉴定与功能表征，该激动剂可优先结合并激活Gαq/11通路。
为筛选可诱导促甲状腺激素受体选择性信号转导的变构调节剂，我们采用了基于转录水平的荧光素酶检测系统：以稳定表达特定应答元件（CRE、NFAT、SRF或SRE）的CHO-TSHR细胞为模型，分别检测Gαs、Gαq/11、Gβγ及Gα12/13偶联所产生的信号。利用该系统，我们发现促甲状腺激素可激活Gαs、Gαq/11及Gα12/13通路，但无法激活Gβγ通路。
我们在0.1 μM、1.0 μM及10 μM三个浓度下对包含5万个化合物的化合物库进行筛选，成功鉴定出一种新型Gq/11通路选择性激动剂（命名为MSq1）：该激动剂可使Gq/11介导的NFAT荧光素酶信号较基线水平提升4倍以上，半数有效浓度（EC50）为8.3×10^-9 M，且仅能微弱诱导Gαs通路及环磷酸腺苷（cyclic adenosine monophosphate, cAMP）的产生。此外，MSq1在化学结构上与所有已报道的促甲状腺激素受体激动剂均存在显著差异。
利用促甲状腺激素受体跨膜结构域（transmembrane domain, TMD）模型进行的分子对接研究显示，MSq1可与跨膜结构域疏水口袋内的螺旋H1、H2、H3及H7，以及细胞外环产生相互作用。当与促甲状腺激素联合给药时，MSq1可呈剂量依赖性抑制促甲状腺激素诱导的甲状腺细胞增殖，但本身并不会对甲状腺细胞的基础增殖水平产生影响。
MSq1的这一意外抑制效应可被蛋白激酶C（protein kinase C, PKC）抑制剂阻断：此时促甲状腺激素诱导的蛋白激酶A（PKA）信号通路抑制被解除，进而恢复细胞增殖能力。由此可见，MSq1的增殖抑制效应源于其能够显著激活PKC，而活化的PKC可进一步抑制由促甲状腺激素受体主要信号通路——cAMP-PKA通路激活所介导的增殖信号。
用MSq1处理大鼠甲状腺细胞（FRTL5）后，关键甲状腺特异性标志物——如甲状腺球蛋白（thyroglobulin, Tg）、甲状腺过氧化物酶（thyroid peroxidase, Tpo）、钠碘同向转运体（sodium iodide symporter, Nis）以及促甲状腺激素受体（Tshr）——的基因表达均未出现上调，进一步表明MSq1及Gαq/11通路的激活并未参与细胞分化过程。
综上，我们鉴定并表征了一种作用于促甲状腺激素受体的新型Gαq/11通路选择性激动剂，该激动剂具备显著的抗增殖活性。因此，对促甲状腺激素受体进行Gq通路偏向性激活，可缓解其正位配体所介导的增殖信号，有望为甲状腺生长调控提供全新的治疗策略。