Studies with mobile rate and binding studies with rat brain homogenates have made it clear that endocrine and various centrally mediated actions of TRH involve uniquely different receptors and that, after decades of effort in various laboratories, we have achieved the separation of these activities with our novel TRH analogues; these analogues all have electronegative groups on the imidazole ring of histidine. Thus, 4(5)- nitro-Im-TRH is highly selective for cardiovascular activity (elevation of heart rate and blood pressure), and may be useful in the treatment of various forms of shock without any endocrine effects. On the other hand, Nva(2)-TRH is a selective analeptic agent without effect on the cardiovascular system; this analogue has served as a research tool for delineation of those binding sites in rat brain which may mediate the analeptic effects of TRH and its other analogues. Computer-assisted structure-activity analysis of a large number of imidazole-substituted analogues has helped us design more selective and more potent members, as well as photoaffinity labels for TRH receptors. Thus, our predictions of the vital role of imidazole-NH tautomer preference in determining selectivity in binding by brain receptors has been validated by the demonstration that 1-Me-5-nitro-Im-TRH binds ca. 3000 times as tightly as 1-Me-4-nitro-Im-TRH. Recently, we have carried out receptor- binding analysis of various TRH analogues with subtle backbone modifications (replacement of the peptide amide with a thioamide surrogate); these studies have allowed us to identify subtle differences in the high affinity TRH receptors in rat pituitary and brain. Current efforts are devoted to the synthesis and evaluation of analogues of TRH designed to meet the requirements of our new concept of """"""""receptor-activated affinity labels"""""""" (RAAL's). Ideally, such a compound would be able to survive the onslaught of nucleophiles in serum and tissues, and yet be able to react selectively and irreversibly with a nucleophile in the appropriate receptor environment.
