The D2 dopamine receptor (DAR) is central in the etiology and/or therapy of many neuropsychiatric disorders. Specifically, D2 DAR antagonism is the hallmark of all FDA-approved antipsychotics and stimulation of the D2 DAR is critical for effective antiparkinsonian therapy. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site within DAR subtypes and between other G protein-coupled receptors. We have developed a high throughput-screening (HTS) platform to interrogate large chemical compound libraries and screened a 370,000+ small molecule library was screened to identify agonists, positive allosteric modulators, or antagonists. The primary HTS assay utilizes a cell line expressing the D2 DAR coupled to a chimeric Gqi5 protein, thereby linking receptor activation to robust Ca2+ mobilization that is measured using a fluorescent readout. We have also developed HTS-formatted secondary assays to measure orthogonal D2 DAR activities (cAMP modulation, beta-arrestin interactions, GIRK channel activation) as well as counter-screening assays to determine selectivity between other DAR subtypes (D1, D3, D4 and D5). Interaction of hit ligands with the orthosteric binding site of the D2 DAR was assessed using standard radioligand binding competition assays. For Hit Compound A, analogs were made to deduce SAR and develop higher affinity analogs. The primary HTS-screen resulted in the identification of 2,288 compounds with agonist activity and 2,294 compounds with antagonist activity. These compounds were cherry-picked to maximize the chances of identifying chemical series with unique activities. Hits were subjected to orthogonal and counter-screening functional assays. While the primary goal for this screen was to identify allosteric compounds, a by-product of this screen and subsequent triaging was the identification of compounds that, while orthosteric, exhibit high selectivity for the D2 DAR and/or are functionally selective with respect to D2 DAR signaling pathways. One such chemotype discovered this way (compound A) selectively activates the D2 DAR in comparison with other DAR subtypes. We found that compound A exhibits full agonist activity with EC50 values ranging from 100 nM to 1 microM using three different functional assays for the D2 DAR;Ca2+ mobilization, inhibition of cAMP accumulation, and beta-arrestin recruitment. Using beta-arrestin recruitment assays to compare with other DARs, we found that compound A has no activity at D1-like DARs (D1 and D5) or on D4 DARs. However, compound A displays either weak partial agonist (<20% of the DA response) or full antagonist activity at D3 DARs. Radioligand binding assays revealed that compound A exhibits Ki values of 1 microM and 100 nM for the D2 and D3 DARs, respectively. Interestingly compound A is a full antagonist with no agonist activity on D2 linked GIRK channel activation. Furthermore, two closely related synthesized analogs of compound A show full D2 agonist activity in G-protein linked D2 functional assays and full antagonist activity in D3 assays (with 10% partial agonism). In summary, compound A is a full and selective agonist at G-protein-linked and arrestin mediated D2 DAR assays, however it shows antagonist activity in D2 GIRK channel assays. Furthermore it also functions as a D3 DAR antagonist on all D3 functional assays tested. This is the first known compound that functionally discriminates between the D2 and D3 DARs and selectively activates the D2 DAR in the absence of D3 DAR activation. The D1 dopamine receptor (DAR) has been implicated in numerous neuropsychiatric disorders, and various D1 ligands have shown great potential as therapeutic agents. However, numerous side effects have limited their clinical utility and it is unknown which D1-mediated signaling pathways are responsible for therapeutic effects versus side effects. Recently it has been appreciated that compounds exist that can modulate one signaling pathway while having no effect on other signaling pathways that are associated with the same receptor. These functionally selective compounds present a unique therapeutic opportunity to target individual pathways while minimizing signaling through others. Previous studies have identified substituted benzazepine compounds, including SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 as D1 agonists. However, the in vivo effects of these compounds have not correlated well to their in vitro pharmacological activities. This is especially true of SKF83959 that has been described as an atypical D1 agonist that does not induce the same behaviors in rodents as typical D1 agonists. A series of substituted benzazepines, and structurally dissimilar D1 agonists, were tested for their functional effects on D1-mediated cAMP accumulation, D1-mediated beta-arrestin recruitment, and D1 receptor internalization using live cell functional assays. With respect to beta-arrestin recruitment, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 were antagonists of this response whereas other substituted benzazepines were nearly full agonists with efficacies ranging from 60-75%. We also examined D1 receptor internalization using a novel beta-galactosidase complementation assay. As with the beta-arrestin recruitment assay, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 exhibited little agonist efficacy, yet the other substituted benzazepines were full agonists in terms of inducing receptor internalization. Our study also used two different D1 cAMP assays: DiscoveRx (DRX) HitHunter, an antibody-based assay that provides a direct measurement of cAMP accumulation, and Codex, which utilizes a cyclic-nucleotide gated channel to measure changes in membrane potential as an indirect readout of cAMP production. In the Codex assay, all compounds elicited an increase in D1 cAMP accumulation similar to that produced by dopamine, with efficacies ranging from 90-110% for the typical agonists and 50-100%for the atypical agonists, effects that were inhibited by the D1 antagonist SCH23390. In contrast, in the DRX D1 HitHunter cAMP assay, the typical agonists displayed a greater variation in agonist efficacy while the substituted benzazepines exhibited a range of efficacies similar to those seen in the Codex assay. Thus, we have identified a group of substituted benzazepine ligands, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670 that are antagonists of D1 receptor-mediated recruitment of beta-arrestin and are devoid of agonist-induced receptor endocytosis. However, in contrast to previous reports, the atypical substituted benzazepines did show full-to-partial agonist activities in two assays of D1-mediated cAMP accumulation. Taken together, these data identify a novel group of substituted benzazepines that are functionally selective for the cAMP-mediated signaling pathway of the D1 receptor. These data may be useful in interpretation of many seemingly contrasting in vivo/in vitro effects of these compounds, and may be useful in further identification of pathway-selective ligands of the D1 dopamine receptor.
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