PATHWAYS TO THYROID STIMULATION: IDENTIFICATION OF NEW POTENT AND SELECTIVE SMALL MOLECULAR AGONISTS TO THE TSH RECEPTOR

Presentation Number: OR28-1
Date of Presentation: June 16th, 2013

Rauf Latif*1, M Rejwan Ali2, Syed A Morshed1, Ting Xu2, Dan F Felsenfeld2, Zerlina Lau3, Mihaly Mezei3 and Terry F Davies4
1Icahn School of Medicine at Mount Sinai & James J Peters VA Medical Center, New York, NY, 2Icahn School of Medicine at Mount Sinai & James J Peters VA Medical Center, 3Icahn School of Medicine at Mount Sinai, New York, NY, 4Mount Sinai School of Medicine and the James J Peters VA Medical Center, New York, NY

Abstract

Novel small molecular ligands (SMLs) to the TSH receptor (TSHR), as first reported by Neumann et al (1), have potential as improved molecular probes and as therapeutic agents for the treatment of thyroid dysfunction including thyroid cancer. To identify additional SMLs to the TSHR we first developed a sensitive and precise transcription-based luciferase-cAMP assay using CHO cells transfected with the human TSHR (HATSHR luci#1). This assay showed cAMP increases of ~1.6 fold with 10μU/ml of bTSH and when adapted and optimized to a 384-shallow well format for high throughput screening (HTS) with 15,000 cells per well had a Z’factor score 0.895 with a CV of 3.78±0.24. We screened 50,000 compounds (ChemBridge) in duplicate using 137 plates at 17μM which resulted in 62 hits using an arbitrary cut-off of mean±3SDs above that of vehicle treated cells with bTSH and the published SML agonist as positive controls.  20 molecules had activity approaching or exceeding that of the agonist and were rescreened against the parent CHO luciferase cell for non-specific activation, against HEK cells transfected with the human LH receptor and against mouse primary Sertoli cells expressing FSH receptors (TM4 line). We selected 5 molecules with good potency and 100% TSHR specificity and the lead molecule (#438) had an impressive EC50 of 2.38 x 10-10 M versus the control SML with an EC50 of 1.601 x10-9 M.  Molecular docking studies using eHiTS, Autodock Vina and AutoDock 4.0 indicated the binding pocket of #438 to be localized to the transmembrane domain with contact residues on ECL2, TM5 and TM6 (see Figure). TM binding specificity was confirmed using a chimeric receptor consisting of an LH receptor ectodomain and TSHR transmembrane (2).  Since the chemical structure of these potent molecules is dissimilar to any reported compounds they have immense potential for in-vivo activity as potent thyroid stimulators.

 

Nothing to Disclose: RL, MRA, SAM, TX, DFF, ZL, MM, TFD