The D1 dopamine receptor has been implicated in numerous neuropsychiatric disorders, and various D1-selective ligands have shown potential as therapeutic agents. Functionally selective, or signaling biased compounds present a unique therapeutic opportunity to target individual pathways while minimizing signaling through others. A series of atypical substituted benzazepines, and D1 typical agonists, were tested for their functional effects on D1-mediated cAMP accumulation, D1-mediated -arrestin recruitment, and D1 receptor internalization using live cell functional assays. With respect to beta-arrestin recruitment, some atypical benzazepines were found to have no agonist efficacy at this response, but instead functioned as antagonists. Other typical agonists exhibited a range of efficacies from 20-110%. As with beta-arrestin recruitment, the atypical benzazepine agonists exhibited little agonist efficacy in an assay of receptor internalization, yet other typical compounds were full agonists. Using two distinct cAMP accumulation assays, typical agonists were found to elicit increases in D1 cAMP accumulation similar to that produced by dopamine, while the atypical agonists were found to be partial-full agonists. Taken together, these experiments identify a novel series of substituted benzazepines that are G protein-biased or functionally selective agonists of the D1 receptor. The D2 dopamine receptor (DAR) is also involved in the etiology and/or therapy of many neuropsychiatric disorders. 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 other G protein-coupled receptors (GPCRs). In order to develop novel molecular scaffolds for the D2 DAR, we used high throughput screening to interrogate a small molecule library and identify hit ligands with distinct functional characteristics, mechanisms of action, and selectivity among DAR subtypes. We have discovered a novel series of small molecule D2 DAR-selective antagonists in this campaign. One such hit was optimized via medicinal chemistry efforts, and a lead compound was identified with high D2 selectivity versus D1, D3, D4 and D5 DARs. Interestingly the binding of this compound to the D2 DAR demonstrated near complete dependence on Na+. Furthermore, the lead compound showed favorable ADME and in vivo pharmacokinetic (PK) properties. In a profiling screen of a large panel of GPCRs the compound showed relatively low cross reactivity. This probe could be a useful pharmacological tool for studying structure activity relationships with the help of computational docking and simulations, as well as an ideal scaffold for the further development of even more highly selective D2 DAR compounds. D2 DAR antagonism is thought to be responsible for the efficacy of antipsychotics, while D3 DAR agonists may be useful as neuroprotective and neurorestorative antiparkinsonian medications. Many antipsychotic drugs belong to the class of 1,4-disubstituted aromatic piperazines and piperidines (1,4-DAPS), although many produce limiting side effects due to the high conservation of the orthosteric binding site among DAR subtypes and other related GPCRs. In order to identify novel small-molecule ligands of DARs with the potential to be highly selective and therefore better drug candidates, our lab has employed a high throughput screening (HTS) approach. Through the NIH Molecular Libraries Program, a novel 1,4 DAP, Compound 3843, was originally identified as a D2 antagonist in a D2-Gqi5 calcium HTS of a 370,000+ small molecule library. Counter-screening assays of beta-arrestin recruitment revealed that this compound selectively activates the D3 DAR and acts as an antagonist at the D2 DAR. Compound 3843 also functions as a D3 DAR agonist in a Go BRET activation assay. We ultimately hope Compound 3843, or its analogs, will be a useful in vitro or in vivo pharmacological tool and chemical optimization efforts are currently underway. The D1 (D1R) and D2 (D2R) dopamine receptors modulate cAMP levels via activation of Gs/olf and Gi/o proteins, but are also are proposed to form hetero-oligomers that, when stimulated, initiate Gq-mediated activation of phospholipase C (PLC) and Ca2+ mobilization. In addition, the compound SKF83959 has been proposed to be a D1-D2 heteromer-selective agonist and has been used as a functional probe of the heteromer in vivo. We investigated several mechanistic and pharmacological aspects of the proposed D1-D2 heteromer and showed that concurrent stimulation of the D1R and D2R were required to elicit Ca2+ mobilization. Additionally, both the long and short isoforms of the D2R were able to form functional heteromers with the D1R. In contrast to previous reports, however, SKF83959 partially inhibited the D1-D2-mediated Ca2+ response and non-selectively exhibited high affinity for a variety of GPCRs. Finally, while over-expression of Gq did boost the D1-D2 mediated Ca2+ response, inhibiting the function of the Gi or Gs proteins, using pertussis toxin or cholera toxin, respectively, drastically reduced the Ca2+ response, as did sequestration of G subunits using catalytically inactive GRK2 mutants. These data indicate that the mechanism of D1-D2-mediated Ca2+ mobilization cannot be solely attributed to Gq protein activation and that additional downstream, crosstalk pathways are involved.

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Xiao, Jingbo; Free, R Benjamin; Barnaeva, Elena et al. (2014) Discovery, optimization, and characterization of novel D2 dopamine receptor selective antagonists. J Med Chem 57:3450-63
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