RECEPTOR-SPECIFIC ACTIVITY OF HETEROMERIC THYROTROPIN (TSH) ANALOGS -DEVELOPMENT OF SYNTHETIC TSH ANTAGONISTS

In all attempt to create potent and specific inhibitors of the interac tion of thyrotropin (thyroid-stimulating hormone [TSH]I with its recep tor ire designed a series of 18 synthetic peptides containing sequence s of both alpha and beta subunits that were shown previously to intera ct with the TSH receptor. These ''heteromeric'' peptide analogs includ ed amino acid residues from alpha 26-46, beta 31-52, beta 88-95 and be ta 101-112 that were arranged variously and Mere seperated each other by artificial amino acid spacers. Each peptide was tested for its abil ity to interact with the TSH receptor in a radio-receptor assay (TSH-R RA) using porcine thyroid membranes and a bio-assay for TSH using FRTL -5 cells. Twelve of the 18 peptides showed binding activity in the TSH -RRA. None of the analogs demonstrated ed thyroid stimulatory activity , but five inhibited TSH bioactivity and were, thus, pure antagonists, the most potent possessing EC(50) values in the 3-5 mu M range. Speci ficity of the antagonists was tested by measuring their ability to inh ibit hCG binding to ovarian membranes, hCG-stimulated progesterone pro duction in MA-10 rat Leydig tumor cells and FSH binding to testicular membranes. Only those peptides that included the alpha-subunit sequenc e CFSR or CCFSR exhibited binding activity for the heterologous recept ors, and that activity was 10-fold lower than ii? the TSH assays. None of the heteromeric peptides showed activity in the hCG bioassays, fur ther demonstrating their specificity as TSH antagonists. These studies illustrate the utility of a synthetic peptide approach in the develop ment of analogs of peptide hormones. Future alterations that significa ntly enhance the potency of these antagonists may results in substance s with clinical efficacy in diseases such as Graves' disease and diffe rentiated thyroid cancer that involve the thyrotropin receptor.