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


METHODS
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%.


CONCLUSIONS
NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.
National Institute of Diabetes and Digestive and Kidney Diseases, Clinical Endocrinology Branch, National Institutes of Health, 50 South Drive, Bethesda, Maryland 20892-8029, USA. SusanneN@intra.niddk.nih.gov
Susanne
S
Neumann
Elena
E
Eliseeva
Joshua G
JG
McCoy
Giorgio
G
Napolitano
Cesidio
C
Giuliani
Fabrizio
F
Monaco
Wenwei
W
Huang
Marvin C
MC
Gershengorn
D000937:Antigen-Antibody Reactions; D002478:Cells, Cultured; D006111:Graves Disease / Q000276:immunology* / Q000473:pathology; D006801:Humans; D007120:Immunochemistry; D018828:Immunoglobulins, Thyroid-Stimulating / Q000096:biosynthesis / Q000378:metabolism*; D007453:Iodide Peroxidase / Q000096:biosynthesis / Q000097:blood; D007457:Iodine Radioisotopes; D011725:Pyridines / Q000138:chemical synthesis / Q000494:pharmacology*; D052999:Quinazolinones / Q000138:chemical synthesis / Q000494:pharmacology*; D011989:Receptors, Thyrotropin / Q000819:agonists* / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D013972:Thyrotropin / Q000097:blood; D013974:Thyroxine / Q000097:blood
D018828:Immunoglobulins, Thyroid-Stimulating; D007457:Iodine Radioisotopes; C556685:NCGC 00229600; D011725:Pyridines; D052999:Quinazolinones; D011989:Receptors, Thyrotropin; D013972:Thyrotropin; D007453:Iodide Peroxidase; D013974:Thyroxine