TY - JOUR T1 - Discovery of a Positive Allosteric Modulator of the TSH Receptor: Potentiation of TSH-mediated Preosteoblast Differentiation In Vitro JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther DO - 10.1124/jpet.117.244095 SP - jpet.117.244095 AU - Susanne Neumann AU - Elena Eliseeva AU - Alisa Boutin AU - Elena Barnaeva AU - Marc Ferrer AU - Noel Southall AU - David Kim AU - Xin Hu AU - Sarah J Morgan AU - Juan J Marugan AU - Marvin C Gershengorn Y1 - 2017/01/01 UR - http://jpet.aspetjournals.org/content/early/2017/10/31/jpet.117.244095.abstract N2 - Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a β-arrestin 1 (β-Arr 1) mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of β-Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated β-Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of β-Arr 1 to the TSHR, but did not activate Gs-mediated signaling pathways, i.e. cAMP production. D3-βArr (NCGC00379308) was selected. In DiscoverX1 cells, D3-βArr stimulated β-Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore, potentiated the effect of TSH in stimulating β-Arr 1 translocation. In U2OS-TSHR cells expressing wild type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3-βArr alone had only a weak effect to upregulate these bone markers, but D3-βArr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the PAM effect of D3-βArr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3-βArr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced β-Arr 1 signaling in osteoblast differentiation. ER -