Abstract

In our previous studies, we demonstrated that the mutation of His393^6.55 to alanine results in an increased affinity of 1,4-disubstituted phenylpiperazines to the dopamine D_2L receptor. This change most likely accounts for the reduced steric hindrance in this part of the binding pocket. In this work, we investigated the role of the steric hindrance imposed by the residue His393^6.55 for the receptor activation modulated by 1,4-DAPs. Site-directed mutagenesis and ligand modifications were used to probe the structural basis of ligand efficacy. The operational model of agonism was employed to quantify the ligand bias between the ability of compounds to inhibit cAMP accumulation and to stimulate ERK1/2 phosphorylation. Whereas substantial ligand biased signaling was observed for the D_2L wild type receptor, an overall increase in agonism was observed for the D_2L His393^6.55Ala mutant without noteworthy functional selectivity. Targeted chemical modification of the phenylpiperazine moiety at the site of its interaction with the residue His393^6.55 led to the functionally selective ligand FAUC350 that has distinct signaling profiles toward adenylyl cyclase and ERK1/2. FAUC350 behaves as an antagonist in the inhibition of cAMP accumulation and as a partial agonist in the stimulation of ERK1/2 phosphorylation (efficacy = 55%). Overall, the residue His393^6.55 and proximate molecular substructures of receptor ligands were identified to be crucial for multidimensional ligand efficacy.