title: New small molecule agonists to the thyrotropin receptor.
abstract: BACKGROUND
  Novel small molecular ligands (SMLs) to the thyrotropin receptor (TSHR) have potential as improved molecular probes and as therapeutic agents for the treatment of thyroid dysfunction and thyroid cancer.
  
  
  METHODS
  To identify novel SMLs to the TSHR, we developed a transcription-based luciferase-cAMP high-throughput screening system and we screened 48,224 compounds from a 100K library in duplicate.
  
  
  RESULTS
  We obtained 62 hits using the cut-off criteria of the mean±three standard deviations above the baseline. Twenty molecules with the greatest activity were rescreened against the parent CHO-luciferase cell for nonspecific activation, and we selected two molecules (MS437 and MS438) with the highest potency for further study. These lead molecules demonstrated no detectible cross-reactivity with homologous receptors when tested against luteinizing hormone (LH)/human chorionic gonadotropin receptor and follicle stimulating hormone receptor-expressing cells. Molecule MS437 had a TSHR-stimulating potency with an EC50 of 13×10(-8) M, and molecule MS438 had an EC50 of 5.3×10(-8) M. The ability of these small molecule agonists to bind to the transmembrane domain of the receptor and initiate signal transduction was suggested by their activation of a chimeric receptor consisting of an LHR ectodomain and a TSHR transmembrane. Molecular modeling demonstrated that these molecules bound to residues S505 and E506 for MS438 and T501 for MS437 in the intrahelical region of transmembrane helix 3. We also examined the G protein activating ability of these molecules using CHO cells co-expressing TSHRs transfected with luciferase reporter vectors in order to measure Gsα, Gβγ, Gαq, and Gα12 activation quantitatively. The MS437 and MS438 molecules showed potent activation of Gsα, Gαq, and Gα12 similar to TSH, but neither the small molecule agonists nor TSH showed activation of the Gβγ pathway. The small molecules MS437 and MS438 also showed upregulation of thyroglobulin (Tg), sodium iodine symporter (NIS), and TSHR gene expression.
  
  
  CONCLUSIONS
  Pharmacokinetic analysis of MS437 and MS438 indicated their pharmacotherapeutic potential, and their intraperitoneal administration to normal female mice resulted in significantly increased serum thyroxine levels, which could be maintained by repeated treatments. These molecules can therefore serve as lead molecules for further development of powerful TSH agonists.
authors.affiliation: 1 Thyroid Research Unit, Department of Medicine, Icahn School of Medicine at Mount Sinai and the James J. Peters VA Medical Center , New York, New York.
authors.forename: Rauf
authors.initials: R
authors.lastname: Latif
authors.forename: M Rejwan
authors.initials: MR
authors.lastname: Ali
authors.forename: Risheng
authors.initials: R
authors.lastname: Ma
authors.forename: Martine
authors.initials: M
authors.lastname: David
authors.forename: Syed A
authors.initials: SA
authors.lastname: Morshed
authors.forename: Michael
authors.initials: M
authors.lastname: Ohlmeyer
authors.forename: Dan P
authors.initials: DP
authors.lastname: Felsenfeld
authors.forename: Zerlina
authors.initials: Z
authors.lastname: Lau
authors.forename: Mihaly
authors.initials: M
authors.lastname: Mezei
authors.forename: Terry F
authors.initials: TF
authors.lastname: Davies
mesh_terms: D000818:Animals; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D005260:Female; D051379:Mice; D011989:Receptors, Thyrotropin / Q000819:agonists*; D013959:Thyroid Diseases / Q000188:drug therapy*
chemical_list: D011989:Receptors, Thyrotropin