G Protein Biased Signaling by Non‐catechol Dopamine D1 Receptor Agonists

Abstract

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 Gs/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 -arrestin indicating that they are G protein biased. The primary goals for this study were to characterize novel signaling by noncatechol agonists and elucidate a mechanism of action for the G protein biased non-catechol agonists. First, the role of -arrestin in D1R agonist-induced endocytosis was established in HEK293 cells that had -arrestin1/2 knocked out by CRISPR/Cas9 genome editing. The knockout of -arrestin1/2 eliminated D1R agonist-induced endocytosis. -arrestin1/2 knockout significantly reduced D1R agonist-induced endocytosis in an ELISA assay that measures cell surface D1R. Furthermore, re-expressing either -arrestin1 or 2 rescued D1R endocytosis in confocal imaging and cell surface ELISA assays. Together, these results indicate that -arrestin1/2 are required for D1R agonist-induced endocytosis. Next, catechol and non-catechol D1R agonists were tested in xi cAMP Glosensor and -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 -arrestin. Interestingly, one non-catechol agonist (PW441) robustly activated both cAMP (Emax = 92%, EC50 = 4.4 nM) and also fully recruited -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 -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 max...

Dopamine D1-like receptor blockade and stimulation decreases operant responding for nicotine and food in male and female rats

“…Furthermore, pre-incubation of rat striatal membranes with A-77636 greatly (76 percent) decreased D1-like receptor binding (Lin et al 1996). A recent study with HEK293 cells showed that A77636 dose-dependently increases cAMP levels and decreases D1-like receptor levels by almost 50 percent (Nilson et al 2020). Importantly, clinical studies indicate that there is a positive relationship between D1-like receptor levels and the subjective effects of drugs, and preclinical studies also show a positive association between D1 receptor levels and the behavioral responses to stimulants (Caine et al 2007;Xu et al 2000;Zhang et al 2000;Zhang et al 2021).…”

“…A study with male hooded rats showed that A77636 decreased food intake by reducing the meal size and duration (Cooper et al 2006). The effect of A77636 on food intake is most likely due to the fact that this drug causes prolonged dopamine D1-like receptor desensitization (Lin et al 1996;Nilson et al 2020).…”

Functionally selective dopamine D1 receptor endocytosis and signaling by catechol and non-catechol agonists

“…Most non-catechol D1R agonists tend to be G protein biased (Gray et al, 2018); however, our study indicates that Cmpd 19 is a balanced agonist, similar to SKF-81297. Notably, the balanced D1R activity of Cmpd 19 described here and orginally by our group (Nilson, 2020;Wang et al, 2019) has also been independently validated and the compound induced robust locomotor activity in a rodent model of Parkinson's disease (see Cmpd 10 in (Martini et al, 2019b)).…”