There are five major types of dopamine receptors, classified into two primary categories, D1-like and D2-like. The D1-like family consists of the D1 and D5 dopamine receptors, whereas, D2-like family consists of the D2, D3, and D4 dopamine receptors. The D2 receptor is important in the etiology and/or therapy of many neuropsychiatric diseases and is an FDA-validated drug target. Unfortunately, truly specific compounds for this receptor have been difficult to obtain due to the highly conserved amino acid sequences that form the orthosteric-binding site, where dopamine and other synthetic drugs bind to the receptor. As a result, drugs that target the D2 receptor frequently interact with other dopamine and non-dopamine receptors leading to unwanted side effects during disease treatment. In order to identify novel D2 receptor-selective compounds, a high throughput screen (HTS) of nearly 400,000 compounds in the NIH-MLPCN library was executed. This effort lead to the identification of a compound (MLS6916) with high affinity and selectivity for the D2 receptor. This compound was subjected to an iterative chemistry approach to improve its drug-like characteristics. Twenty-seven new analogs were synthesized with substituent changes on a separate fragment of the parent molecule. These analogs were studied using a beta-arrestin recruitment assay to determine functional IC50 values for a variety of D2-like receptors including D2, D3, and D4. The parent compound, MLS6916, displayed a 484-fold D2/D3 selectivity, however the compound exhibited only 24-fold D2/D4 selectivity. Of the newly synthesized compounds tested, MLS8039 showed the most promise, with a 1,000-fold D2/D3 selectivity and a 1,200-fold D2/D4 selectivity, indicating that it may be even more D2 selective than the parent scaffold. Further pharmacokinetic screening revealed that, while compound MLS8039 is not as metabolically stable as necessary for drug-like applications, it is very permeable with relatively good solubility. We have established important structure-activity relationships (SAR) around the parent compound and were able to produce more selective analogs of a D2 receptor antagonist. Further chemistry will be completed around the new lead compound MLS8039 with the aim to improve stability and solubility while maintaining its high selectivity. The D2R can activate a spectrum of signaling cascades primarily through G proteins and beta-arrestin recruitment, making it an attractive target for the development of signaling biased ligands. Unlike dopamine, which simultaneously activates G proteins and recruits beta-arrestins, a biased ligand affects only one pathway, and the development of such ligands can allow for a more finely-tuned study of receptor signaling. We previously published the discovery of a highly efficacious, functionally biased D2 dopamine receptor agonist (MLS1547) that can selectively activate G protein signaling while blocking beta-arrestin recruitment. In the interest of understanding the basis for its bias, the signaling properties of 23 MLS1547 analogs were characterized, which ranged from highly biased to unbiased. These results provided the basis for developing structure-activity relationships using pharmacophore modeling and molecular docking analyses. Subsequently, 69 additional MLS1547 analogs were tested for both beta-arrestin and G protein signaling, which refined our model of the structural basis for G protein bias. We have confirmed that a hydrophobic feature, like the chloro group at C5 of MLS1547 is required for G protein-biased signaling. A hydroxyl group para to the C5 group also correlates with strong biased signaling. Replacing the 2-pyridyl group with other nitrogen heterocycles alters the nitrogen basicity, and has a more nuanced effect on bias and signaling strength. Bicyclic scaffolds afforded more potent analogs than the monocyclic scaffolds investigated. Interestingly, some analogs switched from G protein to beta-arrestin bias, opening up additional, interesting avenues for understand the mechanism of biased D2R signaling. This, along with medicinal chemistry approaches will enable a highly targeted approach for investigating D2 receptor signaling in vivo, and may eventually broaden our understanding of pathological D2 receptor signaling in disease states. As an approach to discover highly selective allosteric modulators for the D3 dopamine receptor (DAR), our lab employed high throughput screening technologies. The NIH Molecular Libraries Program 400,000+ small molecule library was initially screened using a D3 DAR-mediated beta-arrestin recruitment assay. Confirmation and counter-screens were performed to obtain an initial assessment of selectivity and mechanisms of action and identified 57 potential negative allosteric modulators (NAMs), 63 potential positive allosteric modulators (PAMs), and 62 potential allosteric agonists. Further triage and characterization identified several D3 DAR-selective putative NAMs, PAMs, and allosteric agonists that are currently being characterized using additional assays to confirm their selectivity, activity, and mechanism of action. As D3-preferring D2/D3 DAR orthosteric agonists show promise as neuroprotective/neurorestorative agents, we conducted preliminary studies using one of the lead allosteric agonists, and found that it displays neuroprotective properties using a cell culture model system. We ultimately hope that these probes will prove useful as in vitro and in vivo pharmacological tools or leads for therapeutic drugs. The D1 dopamine receptor (D1R) has been implicated in numerous neuropsychiatric disorders and various D1R-selective ligands have shown potential as therapeutic agents. In an effort to identify novel selective allosteric modulators of the D1R we recently used a high throughput screening approach. 380,000 small molecules in the NIH Molecular Libraries Screening Center Network (MLPCN) library were screened using a cell line expressing the D1R coupled to G15 resulting in a robust Ca2+ signal upon receptor stimulation. Hit compounds were triaged through secondary functional and radioligand displacement binding assays to determine subtype selectivity and their allosteric versus orthosteric nature. We initially identified 96 putative positive allosteric modulator (PAM) hits that enhanced the EC20 activity of dopamine in the Ca2+ response and 6 of these were subsequently confirmed during triage and were chosen for further characterization. In addition we found approximately 115 agonist and 125 antagonist hits that failed to completely inhibit radioligand binding and thus have been classified as potential allosteric agonists and antagonists or negative allosteric modulators (NAMs). These compounds are currently being characterized using additional assays to confirm thei
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