Small-molecule agonists for the thyrotropin receptor stimulate thyroid function in human thyrocytes and mice

Abstract

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.7TMR ͉ G protein-coupled receptor ͉ low-molecular-weight ligands ͉ radioiodide uptake ͉ TSH receptor S mall-molecule agonists and antagonists for 7-transmembrane-spanning receptors (7TMRs; G protein-coupled receptors) make up approximately 40% of the drugs in clinical use (1). The natural ligands for the 7TMRs for which drugs have been discovered are themselves primarily small molecules, such as norepinephrine, adenosine, and acetylcholine. Smallmolecule ligands for the subclass of 7TMRs for which the natural ligands are 30-kDa heterodimeric glycoprotein hormones have recently begun to be described, but none have been approved for use in humans. For example, small-molecule agonists (2-5) and an antagonist (6) for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR), and small-molecule agonists (7-9) and antagonists (10-12) for the FSH receptor (FSHR) have been reported. The thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) is the third member of the glycoprotein hormone receptor subfamily. We recently reported smallmolecule agonists (13) and an antagonist (14) for human TSHR; however, the agonists were of low affinity and were shown to activate TSHRs only in a model cell system in which human TSHRs were ectopically over-expressed.The physiologic role of TSHRs in the hypothalamic-pituitarythyroid axis is well known. TSH, which is produced in the thyrotrophs of the anterior pituitary gland, is secreted into the circulation in response to TSH-releasing hormone stimulation and in turn stimulates the function of the thyroid follicular cells (i.e., thyrocytes) leading, in particular, to increases in size ...

Thyrotropin Receptor Stimulates Internalization-Independent Persistent Phosphoinositide Signaling

“…We determined whether other TSHR agonists-a small- (Neumann et al, 2009) and a thyroid-stimulating antibody M22 (Sanders et al, 2004)-would cause persistent signaling. We used maximally effective doses of C2 and M22 as determined previously (Sanders et al, 2004;Neumann et al, 2009) and showed that they were maximally effective because the two doses gave similar levels of stimulation.…”

“…We used maximally effective doses of C2 and M22 as determined previously (Sanders et al, 2004;Neumann et al, 2009) and showed that they were maximally effective because the two doses gave similar levels of stimulation. We found qualitatively similar effects of C2 and M22 on persistent cAMP and IP1 signaling.…”

“…The generation of a HEK-EM293 cell lines stably expressing human TSHR (HEK-TSHR cells) or human TRH receptor were described previously (Engel et al, 2006;Neumann et al, 2009). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 10 g/ml streptomycin and 250 g/ml hygromycin B (both from Invitrogen, Carlsbad, CA) at 37°C in a humidified 5% CO 2 incubator.…”

The Antibodies against the Computationally Designed Mimic of the Glycoprotein Hormone Receptor Transmembrane Domain Provide Insights into Receptor Activation and Suppress the Constitutively Activated Receptor Mutants

“…The interhelical network of hydrogen bond between Leu-457, Asp-578, and Asn-619 has been shown to be highly conserved in all the three GpHR members as shown through the rearrangement of carboxylate oxygen of TSHR N674 (Asn-619 in LHR) with Asp-633 (Asp-578 in LHR), resulting in a switch between the activated and inactivated states (29). Control of such an interhelical molecular switch by modulating the ECLs has already been exploited in designing small molecule agonist for TSHR and LHR (19). The above data taken together would suggest that antibody or small molecules binding to ECLs or the exoplasmic face of the TMH can affect global TMD conformation.…”

Targeting thyroid cancer with acid-triggered release of doxorubicin from silicon dioxide nanoparticles

“…[9][10][11][12][13] TSHr can specifically bind the thyroid-stimulating hormone (TSH). This specific binding behavior of TSHr with TSH can be adapted for generating a nanoparticle (NP) drug-delivery system to target thyroid cancer.…”