Structure-activity relationships of adenylate cyclase-coupled beta adrenergic receptors: determination by direct binding studies.

Recently developed techniques for directly studying ligand binding to beta adrenergic receptors with (-)-[3H]alprenolol have been used to delineate in detail the binding specificity of the adenylate cyclase-coupled beta adrenergic receptors in a model system, the frog erythrocyte membrane. The abilities of 60 beta adrenergic agents to compete for the binding sites and to interact with the adenylate cyclase (as agonists or antagonists) were quantitated and compared. The specificity of the receptors determined by direct binding studies or by adenylate cyclase studies was comparable. The KD values of the agents as determined by inhibition of (-)-[3H]alprenolol binding correlated well ( r = 0.95) with their apparent dissociation constants determined by enzyme studies. The latter were determined as the concentrations of agonists necessary to cause 50% maximal enzyme stimulation, or the concentrations of antagonists necessary to produce a 2-fold rightward shift in the (-)-isoproterenol dose-response curve. Agonists and antagonists appeared to compete for the same set of receptor binding sites. Structure-activity relationships determined by the direct binding studies were in excellent agreement with those previously determined in more intact tissue preparations. For agonists the structural features which determined receptor affinity (assessed by direct binding studies) were distinct from those which determined intrinsic activity (maximum ability to stimulate adenylate cyclase). The affinity of agonists was increased by increasing the size of the substituent on the amino nitrogen, by a (-) configuration of the hydroxyl on the β-carbon, and by the presence of a catechol moiety. Methyl or ethyl substitution on the α-carbon had only a slight (generally inhibitory) effect on affinity. Intrinsic activity of agonists was determined primarily by the nature of the substituents on the phenyl ring. Full intrinsic activity requires the presence of hydroxyl groups on the ring at positions 3 and 4 as well as the β-carbon hydroxyl in the (-) configuration. Deletion of the β-carbon hydroxyl, as in compounds such as dopamine, dobutamine, and related agents, leads to substantial loss of intrinsic activity and affinity even in the presence of large amino nitrogen substituents. A methanesulfonamide group substituted for the hydroxyl in position 3 on the ring results in reduced intrinsic activity. Deletion of the ring hydroxyl at either position 3 or 4 or substitution by chlorine produces competitive antagonists. Structure-activity relationships of antagonists were similar to those of agonists, except that the catechol moiety was replaced by a single or double aromatic ring structure. Separation of this moiety from the ethanolamine side chain by an ether function significantly increased affinity. When a phenyl group was present, a single substituent at the para position was associated with reduced affinity. ACKNOWLEDGMENT We gratefully acknowledge the expert assistance of the analytical chemistry department of New England Nuclear Corporation in developing chromatographic procedures for (-)-alprenolol.