Molecular And Pharmacological Studies Of Dopamine Receptors

National Institute Of Neurological Disorders And Stroke

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Abstract

The D1 dopamine receptor (D1R) is a G protein-coupled receptor that signals through activating adenylyl cyclase and raising intracellular cAMP levels. When activated, the D1R also recruits the scaffolding protein beta-arrestin, which promotes receptor desensitization and internalization, as well as additional downstream signaling pathways. These processes are triggered through receptor phosphorylation by G protein-coupled receptor kinases (GRKs), although the precise phosphorylation sites and their role in recruiting beta-arrestin to the D1R remains incompletely described. In this study, we have used detailed mutational and in situ phosphorylation analyses to completely identify the GRK-mediated phosphorylation sites on the D1R. Our results indicate that GRKs can phosphorylate 14 serine and threonine residues within the C-terminus and the third in-tracellular loop (ICL3) of the receptor, and that this occurs in a hierarchical fashion where phosphorylation of the C-terminus precedes that of the ICL3. Using beta-arrestin recruitment assays, we identified a cluster of phosphorylation sites in the proximal region of the C-terminus that drive beta-arrestin binding to the D1R. We further provide evidence that phosphorylation sites in the ICL3 are responsible for beta-arrestin activation leading to receptor internalization. Our results suggest that distinct D1R GRK phosphorylation sites are involved in beta-arrestin binding and activation.. Schizophrenia is a devastating neuropsychiatric illness impacting approximately 1% of the global population and is the 15th leading cause of disability worldwide. Current therapies treat mostly the positive symptoms and are associated with a plethora of off-target side effects such as sedation, weight gain, and diabetes, among others. All FDA-approved antipsychotic medications target the D2 dopamine receptor (D2R) but also exhibit polypharmacology with other receptors. Further, there are few D2R antagonists that can selectively inhibit the D2R without also antagonizing the D3 and D4 dopamine receptors (D3R, D4R, respectively). We recently identified a D2R-selective antagonist scaffold, MLS6916, from a high throughput screen of the D2R. When counter-screened against 168 GPCRs using -arrestin recruitment as a functional readout, 10 uM MLS6916 only inhibited the D2R, and to a lesser extent, the D4R. Further, using radioligand binding competition assays, MLS6916 was >200-fold D2R>D3R selective and 12-fold D2R>D4R selective. Despite its promising D2R selectivity, MLS6916 was found to exhibit poor metabolic stability when assayed using rat liver microsomes. Interestingly, we found high species variability with respect to metabolic stability using rat, mouse, and human liver microsomes. While many analogs exhibited poor metabolic stability in rat liver microsomes, we observed equal or worse stability in mouse liver microsomes, but dramatically higher stability in human liver microsomes. High metabolic stability in humans is essential for moving a compound into the clinic, but preclinical studies will require at least moderate metabolic stability in rodents to employ animal models that are predictive of antipsychotic efficacy and/or adverse side effects. Thus, to chemically optimize this scaffold and explore its structure-activity relationships, greater than 100 analogs were synthesized to identify modifications that might result in improved metabolic stability. All analogs were also evaluated for D2R, D3R, and D4R activities using radioligand binding competition and beta-arrestin recruitment assays. Lead compounds were identified that possessed D2R Ki values of <100 nM, were highly selective versus the D3R and D4, and exhibited improved metabolic stability in mouse and/or human liver microsomes. We further determined the pharmacokinetic profiles of the most promising compounds in mice since this species has good models for predicting antipsychotic efficacy. After injecting 30 mg/kg i.p., we found that the compounds exhibited t1/2 values of 5-6 hr in both plasma and brain. Importantly, the compounds exhibited 1:1 brain-plasma ratios with Cmax values >10 uM indicating excellent brain penetration. In summary, we have identified lead candidate compounds that have exceptional D2R-selectivity, excellent metabolic stabilities in human liver microsomes, and sufficient metabolic stability in mice to conduct behavioral studies. Future studies will investigate preclinical antipsychotic efficacy in mouse models as well as the potential for on-target side effects such as catalepsy. While antagonists of D2Rs are currently used as antipsychotics, D3R-selective antagonists might served as therapeutics for substance use disorder (SUD) as they could attenuate drug craving symptoms without the motor side effects produced by D2R antagonists. However, discovery of D3R-selective compounds is challenging due to high sequence homology of the D2R and D3R within their orthosteric binding sites, leading to the potential for off-target side effects produced by currently available compounds due to simultaneous antagonism of both subtypes or other closely related receptors. Our lab has endeavored to overcome the selectivity challenges posed by orthosteric antagonists by utilizing a D3R-mediated beta-arrestin recruitment assay to screen the NIH Molecular Libraries Program 400,000+ small molecule library for compounds that inhibit the D3R via binding to less conserved allosteric sites. The most potent hit compound, MLS6357, was selective for the D3R versus the D2R and D4R in several functional outputs including beta-arrestin recruitment and G-protein activation. Radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross-reactivity with other GPCRs. Additionally, Schild-type functional assays showed that MLS6357 acts as a purely non-competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized >70 analogs of MLS6357 using iterative medicinal chemistry approaches which produced analogs that are 100-fold and 60-fold more potent than the parent compound in D3R-mediated beta-arrestin recruitment and G-protein activation assays, respectively, and revealed structure-activity relationships for further optimization of the scaffold. Moreover, some analogs appear to display functional selectivity for inhibition of G-protein activation versus inhibition of beta-arrestin recruitment, and vice versa, and some also display inverse agonist activity in G-protein signaling assays. Using in vivo pharmacokinetic experiments in mice via i.p. administration, one of the lead analogs was found to be brain penetrant and achieved sufficient concentrations to occupy the D3R in vivo. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R-related pathophysiology.