Dual Agonistic and Antagonistic Property of Nonpeptide Angiotensin AT₁Ligands: Susceptibility to Receptor Mutations

Abstract

Two nonpeptide ligands that differ chemically by only a single methyl group but have agonistic (L-162,782) and antagonistic (L-162,389) properties in vivo were characterized on the cloned angiotensin AT1 receptor. Both compounds bound with high affinity (K(I) = 8 and 28 nM, respectively) to the AT1 receptor expressed transiently in COS-7 cells as determined in radioligand competition assays. L-162,782 acted as a powerful partial agonist, stimulating phosphatidylinositol turnover with a bell-shaped dose-response curve to 64% of the maximal level reached in response to angiotensin II. Surprisingly, L-162,389 also stimulated phosphatidylinositol turnover, albeit only to a small percentage of the angiotensin response. The prototype nonpeptide AT1 agonist L-162,313 gave a response of approximately 50%. The apparent EC50 values for all three compounds in stimulating phosphatidylinositol turnover were similar, approximately 30 nM, corresponding to their binding affinity. Each of the three compounds also acted as angiotensin antagonists, yet in this capacity the compounds differed markedly, with IC50 values ranging from 1.05 x 10(-7) M for L-162,389 to 6.5 x 10(-6) for L-162,782. A series of point mutations in the transmembrane segments (TMs) of the AT1 receptor had only minor effect on the binding affinity of the nonpeptide compounds, with the exception of A104V at the top of TM III, which selectively impaired the binding of L-162,782 and L-162,389. Substitutions in the middle of TM III, VI, or VII, which did not affect the binding affinity of the compounds, impaired or eliminated the agonistic efficacy of the nonpeptides but with only minor or no effect on the angiotensin potency or efficacy. Thus, in the N295D rat AT1 construct, L-162,782, L-162,313, and L-162,389 all antagonized the angiotensin-induced phosphatidylinositol turnover with surprisingly similar IC50 values (90-180 nM), and they all bound with unaltered, high affinity (22-36 nM). However, L-162,313 and L-162,782 could stimulate phosphatidylinositol turnover to only 20% of that of angiotensin. It is concluded that minor chemical modifications of either the compound or the receptor can dramatically alter the agonistic efficacy of biphenyl imidazole compounds on the AT1 receptor without affecting their affinity, as determined in binding assays, and that a number of substitutions in the middle of the TM segments affect the efficacy of nonpeptide agonists as opposed to angiotensin.

Identification of a series of CCK-2 receptor nonpeptide agonists: Sensitivity to stereochemistry and a receptor point mutation

“…There is an increasing number of studies reporting that slight modifications in ligand structure may translate into loss or gain of agonist function (25)(26)(27)(28)(29)(30). At the wild-type CCK-2R, the addition of two methyl groups to the S enantiomer of compound 3 markedly enhances ligand function (compare 2S and 3S in Table 1 and Fig.…”

“…In a CCK-1 receptor ligand derived from a 1,5-benzodiazepine template, the addition of a single methyl group converts an antagonist (11b) to a relatively strong partial agonist (11c) that has Ϸ66% of the activity of the endogenous hormone, CCK-8 (26). Similarly, at the angiotensin AT1 receptor, a biphenyl-based nonpeptide antagonist can be converted into an agonist by the addition of a single methyl group (27).…”

Ghrelin Receptor Inverse Agonists: Identification of an Active Peptide Core and Its Interaction Epitopes on the Receptor

“…In the latter case, the compound is then either a neutral antagonist or it may even have gained agonist efficacy and may be "balancing between" agonism and inverse agonism. It should be noted that small chemical changes in nonpeptide agonists can, in certain receptor systems, change them into antagonists (Perlman et al, 1997).…”

Biased Ligands for Better Cardiovascular Drugs

“…65,66 Striking differences in the residues contributing to agonist and antagonist peptide binding and efficacy support the expectation that ligand binding stabilizes or destabilizes particular receptor structural features, and that integration of these interactions permit or restrict AT1R signal-coupling mechanisms. 90,91 Inconsistent with a simpler "lock and key" model of agonist and antagonist efficacy. Intriguingly, these findings have been extended by homology modeling of the AT1R to crystal structures of rhodopsin, ␤2 adrenergic, and adenosine A2 receptors to hypothesize several key residues that may be engaged in ␤-arrestin coupling and the difference between balanced and ␤-arrestin-biased AT1R ligands.…”