Development of pyrimidone D1 dopamine receptor positive allosteric modulators.

MLS1082 is a structurally novel pyrimidone-based D1-like dopamine receptor positive allosteric modulator. Potentiation of D1 dopamine receptor (D1R) signaling is a therapeutic strategy for treating neurocognitive disorders. Here, we investigate the relationship between D1R potentiation and two prominent structural features of MLS1082, namely the pendant N-aryl and C-alkyl groups on the pyrimidone ring. To this end, we synthesized 24 new analogues and characterized their ability to potentiate dopamine signaling at the D1R and the closely related D5R. We identified structure-activity relationship trends for both aryl and alkyl modifications and our efforts afforded several analogues with improvements in activity. The most effective analogues demonstrated an approximately 8-fold amplification of dopamine-mediated D1R signaling. These findings advance the understanding of structural moieties underlying the activity of pyrimidone-based D1R positive allosteric modulators.

Identification of Positive Allosteric Modulators of the D1 Dopamine Receptor That Act at Diverse Binding Sites.

The D1 dopamine receptor is linked to a variety of neuropsychiatric disorders and represents an attractive drug target for the enhancement of cognition in schizophrenia, Alzheimer disease, and other disorders. Positive allosteric modulators (PAMs), with their potential for greater selectivity and larger therapeutic windows, may represent a viable drug development strategy, as orthosteric D1 receptor agonists possess known clinical liabilities. We discovered two structurally distinct D1 receptor PAMs, MLS6585 and MLS1082, via a high-throughput screen of the NIH Molecular Libraries program small-molecule library. Both compounds potentiate dopamine-stimulated G protein- and ß-arrestin-mediated signaling and increase the affinity of dopamine for the D1 receptor with low micromolar potencies. Neither compound displayed any intrinsic agonist activity. Both compounds were also found to potentiate the efficacy of partial agonists. We tested maximally effective concentrations of each PAM in combination to determine if the compounds might act at separate or similar sites. In combination, MLS1082 + MLS6585 produced an additive potentiation of dopamine potency beyond that caused by either PAM alone for both ß-arrestin recruitment and cAMP accumulation, suggesting diverse sites of action. In addition, MLS6585, but not MLS1082, had additive activity with the previously described D1 receptor PAM "Compound B," suggesting that MLS1082 and Compound B may share a common binding site. A point mutation (R130Q) in the D1 receptor was found to abrogate MLS1082 activity without affecting that of MLS6585, suggesting this residue may be involved in the binding/activity of MLS1082 but not that of MLS6585. Together, MLS1082 and MLS6585 may serve as important tool compounds for the characterization of diverse allosteric sites on the D1 receptor as well as the development of optimized lead compounds for therapeutic use.

PNA-Based Multivalent Scaffolds Activate the Dopamine D2 Receptor.

Peptide nucleic acid scaffolds represent a promising tool to interrogate the multivalent effects of ligand binding to a membrane receptor. Dopamine D2 receptors (D2R) are a class of G-protein coupled receptors (GPCRs), and the formation of higher-ordered structures of these receptors has been associated with the progression of several neurological diseases. In this Letter, we describe the synthesis of a library of ligand-modified PNAs bearing a known D2R agonist, (±)-PPHT. The D2R activity for each construct was assessed, and the multivalent effects were evaluated.

Identification of G protein-biased agonists that fail to recruit ß-arrestin or promote internalization of the D1 dopamine receptor.

The D1 dopamine receptor (D1R) has been implicated in numerous neuropsychiatric disorders, and D1R-selective ligands have potential as therapeutic agents. Previous studies have identified substituted benzazepines as D1R-selective agonists, but the in vivo effects of these compounds have not correlated well with their in vitro pharmacological activities. A series of substituted benzazepines, and structurally dissimilar D1R-selective agonists, were tested for their functional effects on D1R-mediated cAMP accumulation, D1R-promoted ß-arrestin recruitment, and D1R internalization using live cell functional assays. All compounds tested elicited an increase in the level of cAMP accumulation, albeit with a range of efficacies. However, when the compounds were evaluated for ß-arrestin recruitment, a subset of substituted benzazepines, SKF83959, SKF38393, SKF82957, SKF77434, and SKF75670, failed to activate this pathway, whereas the others showed similar activation efficacies as seen with cAMP accumulation. When tested as antagonists, the five biased compounds all inhibited dopamine-stimulated ß-arrestin recruitment. Further, D1R internalization assays revealed a corroborating pattern of activity in that the G protein-biased compounds failed to promote D1R internalization. Interestingly, the biased signaling was unique for the D1R, as the same compounds were agonists of the related D5 dopamine receptor (D5R), but revealed no signaling bias. We have identified a group of substituted benzazepine ligands that are agonists at D1R-mediated G protein signaling, but antagonists of D1R recruitment of ß-arrestin, and also devoid of agonist-induced receptor endocytosis. These data may be useful for interpreting the contrasting effects of these compounds in vitro versus in vivo, and also for the understanding of pathway-selective signaling of the D1R.

(-)-Stepholidine is a potent pan-dopamine receptor antagonist of both G protein- and ß-arrestin-mediated signaling.

RATIONALE: (-)-Stepholidine is a tetrahydroberberine alkaloid that is known to interact with dopamine receptors and has also been proposed as a novel antipsychotic agent. Its suggested novelty lies in the fact that it has been proposed to have D1-like receptor agonist and D2-like receptor antagonist properties. Thus, it might be effective in treating both positive and negative (cognition) symptoms of schizophrenia. However, its activity on specific dopamine receptor subtypes has not been clarified, especially with respect to its ability to activate D1-like receptors. OBJECTIVES: We wished to examine the affinity and functional activity of (-)-stepholidine at each of the human dopamine receptor subtypes expressed in a defined cellular environment. METHODS: D1-D5 dopamine receptors were stably expressed in cell lines and their interactions with (-)-stepholidine were examined using radioligand binding and various functional signaling assays. Radioligand binding assays were also performed using bovine striatal membranes. RESULTS: (-)-Stepholidine exhibited high (nM) affinity for D1 and D5 receptors, somewhat lower (two- to four-fold) affinity for D2 and D3 receptors, and low micromolar affinity for D4 receptors. Functionally, (-)-stepholidine was ineffective in activating G protein-mediated signaling of D1-like and D2 receptors and was also ineffective in stimulating ß-arrestin recruitment to any dopamine receptor subtype. It did, however, antagonize all of these responses. It also antagonized D1-D2 heteromer-mediated Ca(2+) mobilization. Radioligand binding assays of D1-like receptors in brain membranes also indicated that (-)-stepholidine binds to the D1 receptor with antagonist-like properties. CONCLUSIONS: (-)-Stepholidine is a pan-dopamine receptor antagonist and its in vivo effects are largely mediated through dopamine receptor blockade with potential cross-talk to other receptors or signaling proteins.

Discovery and characterization of a G protein-biased agonist that inhibits ß-arrestin recruitment to the D2 dopamine receptor.

A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and ß-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate ß-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit ß-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated ß-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate ß-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate ß-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.

Discovery, optimization, and characterization of novel D2 dopamine receptor selective antagonists.

The D2 dopamine receptor (D2 DAR) is one of the most validated drug targets for neuropsychiatric and endocrine disorders. However, clinically approved drugs targeting D2 DAR display poor selectivity between the D2 and other receptors, especially the D3 DAR. This lack of selectivity may lead to undesirable side effects. Here we describe the chemical and pharmacological characterization of a novel D2 DAR antagonist series with excellent D2 versus D1, D3, D4, and D5 receptor selectivity. The final probe 65 was obtained through a quantitative high-throughput screening campaign, followed by medicinal chemistry optimization, to yield a selective molecule with good in vitro physical properties, metabolic stability, and in vivo pharmacokinetics. The optimized molecule may be a useful in vivo probe for studying D2 DAR signal modulation and could also serve as a lead compound for the development of D2 DAR-selective druglike molecules for the treatment of multiple neuropsychiatric and endocrine disorders.

CC12, a P450/epoxygenase inhibitor, acts in the rat rostral, ventromedial medulla to attenuate morphine antinociception.

Brain cytochrome P450 epoxygenases were recently shown to play an essential role in mediating the pain-relieving properties of morphine. To identify the CNS sites containing the morphine-relevant P450s, the effects of intracerebral (ic) microinjections of the P450 inhibitor CC12 were determined on morphine antinociception in rats. CC12 inhibited morphine antinociception when both drugs were injected into the rostral ventromedial medulla (RVM), but not following co-injections into the periaqueductal gray (PAG) or into the spinal subarachnoid space. In addition, intra-RVM CC12 pretreatment nearly completely blocked the effects of morphine following intracerebroventricular (icv) administration. Although morphine is thought to act in both the PAG and RVM by pre-synaptic inhibition of inhibitory GABAergic transmission, the present findings show that 1) the mechanism of morphine action differs between these two brainstem areas, and 2) P450 activity within the RVM is important for supraspinal morphine antinociception. Characterization of morphine-P450 interactions within RVM circuits will further enhance the understanding of the biochemistry of pain relief.

Modification of cocaine self-administration by buspirone (buspar®): potential involvement of D3 and D4 dopamine receptors.

Converging lines of evidence indicate that elevations in synaptic dopamine levels play a pivotal role in the reinforcing effects of cocaine, which are associated with its abuse liability. This evidence has led to the exploration of dopamine receptor blockers as pharmacotherapy for cocaine addiction. While neither D1 nor D2 receptor antagonists have proven effective, medications acting at two other potential targets, D3 and D4 receptors, have yet to be explored for this indication in the clinic. Buspirone, a 5-HT1A partial agonist approved for the treatment of anxiety, has been reported to also bind with high affinity to D3 and D4 receptors. In view of this biochemical profile, the present research was conducted to examine both the functional effects of buspirone on these receptors and, in non-human primates, its ability to modify the reinforcing effects of i.v. cocaine in a behaviourally selective manner. Radioligand binding studies confirmed that buspirone binds with high affinity to recombinant human D3 and D4 receptors (∼98 and ∼29 nm respectively). Live cell functional assays also revealed that buspirone, and its metabolites, function as antagonists at both D3 and D4 receptors. In behavioural studies, doses of buspirone that had inconsistent effects on food-maintained responding (0.1 or 0.3 mg/kg i.m.) produced a marked downward shift in the dose-effect function for cocaine-maintained behaviour, reflecting substantial decreases in self-administration of one or more unit doses of i.v. cocaine in each subject. These results support the further evaluation of buspirone as a candidate medication for the management of cocaine addiction.

Brain P450 epoxygenase activity is required for the antinociceptive effects of improgan, a nonopioid analgesic.

The search for the mechanism of action of improgan (a nonopioid analgesic) led to the recent discovery of CC12, a compound that blocks improgan antinociception. Because CC12 is a cytochrome P450 inhibitor, and brain P450 mechanisms were recently shown to be required in opioid analgesic signaling, pharmacological and transgenic studies were performed in rodents to test the hypothesis that improgan antinociception requires brain P450 epoxygenase activity. Intracerebroventricular (i.c.v.) administration of the P450 inhibitors miconazole and fluconazole, and the arachidonic acid (AA) epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH) potently inhibited improgan antinociception in rats at doses that were inactive alone. MW06-25, a new P450 inhibitor that combines chemical features of CC12 and miconazole, also potently blocked improgan antinociception. Although miconazole and CC12 were weakly active at opioid and histamine H(3) receptors, MW06-25 showed no activity at these sites, yet retained potent P450-inhibiting properties. The P450 hypothesis was also tested in Cpr(low) mice, a viable knock-in model with dramatically reduced brain P450 activity. Improgan (145 nmol, i.c.v.) antinociception was reduced by 37% to 59% in Cpr(low) mice, as compared with control mice. Moreover, CC12 pretreatment (200 nmol, i.c.v.) abolished improgan action (70% to 91%) in control mice, but had no significant effect in Cpr(low) mice. Thus, improgan's activation of bulbospinal nonopioid analgesic circuits requires brain P450 epoxygenase activity. A model is proposed in which (1) improgan activates an unknown receptor to trigger downstream P450 activity, and (2) brainstem epoxygenase activity is a point of convergence for opioid and nonopioid analgesic signaling.

Opioids activate brain analgesic circuits through cytochrome P450/epoxygenase signaling.

To assess the importance of brain cytochrome P450 (P450) activity in mu opioid analgesic action, we generated a mutant mouse with brain neuron-specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine.