G protein biased signaling by non-catechol dopamine D1 receptor agonists

Dopamine is a catecholamine neurotransmitter with essential roles in voluntary movement, working memory, attention, and reward. Dopamine acts through five G protein coupled receptors with the D1 and D5 receptors (D1R) stimulating Galphas/olf activation and increasing neuronal excitability. Deficits in D1R signaling are implicated in Parkinson’s disease motor deficits as well as cognitive deficits in schizophrenia and attention deficit hyperactivity disorder. For more than 40 years, academic and industry scientists have been searching for a drug-like D1R agonist, but this has remained elusive. The challenge in developing D1R selective agonists is that all previous agonists possess a common problematic chemical moiety, a catechol. Catechols are associated with poor oral bioavailability, poor brain penetration, and rapid metabolism in the serum. Very recently, the breakthrough discovery of the first non-catechol D1R selective agonists overcame the pitfalls associated with the catechols. Unexpectedly, the non-catechol agonists also selectively activate G protein signaling without engaging beta-arrestin indicating that they are G protein biased. The primary goals for this study were to characterize novel signaling by non-catechol agonists and elucidate a mechanism of action for the G protein biased non-catechol agonists. First, the role of beta-arrestin in D1R agonist-induced endocytosis was established in HEK293 cells that had beta-arrestin1/2 knocked out by CRISPR/Cas9 genome editing. The knockout of beta-arrestin1/2 eliminated D1R agonist-induced endocytosis. beta-arrestin1/2 knockout significantly reduced D1R agonist-induced endocytosis in an ELISA assay that measures cell surface D1R. Furthermore, re-expressing either beta-arrestin1 or 2 rescued D1R endocytosis in confocal imaging and cell surface ELISA assays. Together, these results indicate that beta-arrestin1/2 are required for D1R agonist-induced endocytosis. Next, catechol and non-catechol D1R agonists were tested in cAMP Glosensor and beta-arrestin Tango assays to investigate potential biased signaling. The unbiased catechol D1R full agonist SKF-81297 was used as the reference compound in all following studies. The non-catechol D1R agonists dose-dependently increased cAMP production in HEK293 cells similar to the full agonist SKF-81297 (Emax 100%), but did not engage beta-arrestin. Interestingly, one non-catechol agonist (PW441) robustly activated both cAMP (Emax = 92%, EC50 = 4.4 nM) and also fully recruited beta-arrestin (Emax = 100%, EC50 = 100 nM). The catechol agonist A-77636 dose-dependently increased full cAMP production (Emax = 104%, EC50 = 3.1 nM) but was a super agonist for beta-arrestin recruitment (Emax = 130%, EC50 = 35 nM). To determine the effect of G protein biased agonists on D1R endocytosis, the catechol and non-catechol D1R agonists were tested in imaging and cell surface ELISA assays. The non-catechol G protein biased agonists all induced significantly less total D1R endocytosis than the catechol agonist SKF-81297. The pure G protein biased agonists PF-1119 and PW464 maximally induced 5% and 11% loss of cell surface D1R, respectively. In contrast, the catechol A-77636 maximally induced 47% loss of cell surface D1R and induced significantly more total endocytosis than SKF-81297. Moreover, the efficacy for beta-arrestin recruitment strongly correlates to the maximum receptor endocytosis in Spearman’s correlation analysis (r = 0.96, p<0.05). Collectively, this study demonstrates the essential role of beta-arrestin in D1R agonist-induced endocytosis and characterizes novel non-catechol agonists. In addition, the discovery of the first balanced non-catechol D1R selective agonist adds a unique tool for future in vitro and in vivo studies. These results further elucidate a mechanism of action for the G protein biased non-catechol agonists in which agonist binding induces G protein activation without also inducing D1R endocytosis. These results provide insights into the molecular mechanism of the G protein biased non-catechol agonists. While the clinical efficacy of the non-catechol agonists is currently being explored, the mechanism of action is not fully understood. This study explored novel derivatives and their downstream effects on D1R endocytosis.