A new small-molecule antagonist inhibits Graves' disease antibody activation of the TSH receptor.

Methods Development
Medicinal Chemistry


CONTEXT: Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. OBJECTIVE: Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. DESIGN: We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. RESULTS: We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. CONCLUSION: NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.


Neumann, Susanne; Eliseeva, Elena; McCoy, Joshua G; Napolitano, Giorgio; Giuliani, Cesidio; Monaco, Fabrizio; Huang, Wenwei; Gershengorn, Marvin C;


  • Antigen-Antibody Reactions
  • Cells, Cultured
  • Graves Disease/ immunology
  • Graves Disease/ pathology
  • Humans
  • Immunochemistry
  • Immunoglobulins, Thyroid-Stimulating/ biosynthesis
  • Immunoglobulins, Thyroid-Stimulating/ metabolism
  • Iodide Peroxidase/ biosynthesis
  • Iodide Peroxidase/ blood
  • Iodine Radioisotopes
  • Pyridines/ chemical synthesis
  • Pyridines/ pharmacology
  • Quinazolinones/ chemical synthesis
  • Quinazolinones/ pharmacology
  • Receptors, Thyrotropin/ agonists
  • Receptors, Thyrotropin/ biosynthesis
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction/ physiology
  • Thyroid Gland/ drug effects
  • Thyroid Gland/ metabolism
  • Thyroid Gland/ pathology
  • Thyrotropin/ blood
  • Thyroxine/ blood

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