Discovery and characterization of benzyloxy piperidine based dopamine 4 receptor antagonists.

The dopamine receptor 4 (D4R) is highly expressed in both motor, associative and limbic subdivisions of the cortico-basal ganglia network. Due to the distribution in the brain, there is mounting evidence pointing to a role for the D4R in the modulation of this network and its subsequent involvement in l-DOPA induced dyskinesias in Parkinson's disease. As part of our continued effort in the discovery of novel D4R antagonists, we report the discovery and characterization of a new 3- or 4-benzyloxypiperidine scaffold as D4R antagonists. We report several D4R selective compounds (>30-fold vs. other dopamine receptor subtypes) with improved in vitro and in vivo stability over previously reported D4R antagonists.

Recent findings leading to the discovery of selective dopamine D4 receptor ligands for the treatment of widespread diseases.

Since its discovery, the dopamine D4 receptor (D4R) has been suggested to be an attractive target for the treatment of neuropsychiatric diseases. Novel findings have renewed the interest in such a receptor as an emerging target for the management of different diseases, including cancer, Parkinson's disease, alcohol or substance use disorders, eating disorders, erectile dysfunction and cognitive deficits. The recently resolved crystal structures of D4R in complexes with the potent ligands nemonapride and L-745870 strongly improved the knowledge on the molecular mechanisms involving the D4R functions and may help medicinal chemists in drug design. This review is focused on the recent development of the subtype selective D4R ligands belonging to classical or new chemotypes. Moreover, ligands showing functional selectivity toward G protein activation or ß-arrestin recruitment and the effects of selective D4R ligands on the above-mentioned diseases are discussed.

Lack of dopamine D4 receptor participation in mouse hyperdopaminergic locomotor response.

Chronic methamphetamine (METH) treatment induces behavioral sensitization in rodents. During this process, hyperactivation of the mesolimbic dopamine system plays a central role, and dopamine D2-like receptor-based antipsychotics are known to alleviate the behavioral hyperactivity. The atypical antipsychotic, clozapine (Clz), acts partially as a dopamine D4 receptor (D4R) antagonist and mitigates hyperdopaminergic drug addiction and/or comorbid psychotic symptoms; however, it remains unclear whether D4R blockade contributes to the therapeutic effects of Clz. Here, we evaluated the potential role of D4R in regulating hyperdopaminergia-induced behavioral hyperactivity in METH behavioral sensitization and dopamine transporter (DAT) knockdown (KD) mice. Clz or a D4R-selective antagonist, L-745,870, were co-administered to mice with daily METH in a METH sensitization model, and Clz or L-745,870 were administered alone in a DAT KD hyperactivity model. Locomotor activity and accumbal D4R expression were analyzed. Clz suppressed both the initiation and expression of METH behavioral sensitization, as well as DAT KD hyperactivity. However, repetitive Clz treatment induced tolerance to the suppression effect on METH sensitization initiation. In contrast, D4R inhibition by L-745,870 had no effect on METH sensitization or DAT KD hyperactivity. Accumbal D4R expression was similar between METH-sensitized mice with and without Clz co-treatment. In sum, our results suggest the mesolimbic D4R does not participate in behavioral sensitization encoded by hyperdopaminergia, a finding which likely extends to the therapeutic effects of Clz. Therefore, molecular targets other than D4R should be prioritized in the development of future therapeutics for treatment of hyperdopaminergia-dependent neuropsychiatric disorders.

Luteolin, a Potent Human Monoamine Oxidase-A Inhibitor and Dopamine D4 and Vasopressin V1A Receptor Antagonist.

Luteolin, a flavonoid widely distributed in the plant kingdom, contains two benzene rings and hydroxyl groups, and this structural specificity contributes to its diverse biological activities. However, no previous studies have simultaneously investigated the therapeutic potency of luteolin isolated from a plant as an antipsychotic and antidepressant. Here, luteolin exhibited selective inhibition of hMAO-A (IC50 = 8.57 ± 0.47 µM) over hMAO-B (IC50 > 100 µM). In silico proteochemometric modeling predicted promising targets of luteolin, and verification via cell-based G protein-coupled receptor functional assays showed that luteolin is a selective antagonist of the vasopressin receptor V1AR (IC50 = 19.49 ± 6.32 µM) and the dopamine D4 receptor (IC50 = 39.59 ± 1.46 µM). Molecular docking showed the tight binding of luteolin with a low binding score and the high stability of the luteolin-receptor complex, corroborating its functional effect. Thus, hMAO-A, hD4R, and hV1AR are prime targets of luteolin and potential alternatives for the management of neurodegenerative diseases.

Dopamine D4 receptors in the lateral habenula regulate depression-related behaviors via a pre-synaptic mechanism in experimental Parkinson's disease.

Although multiple studies report that unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNc) in rats induce depressive-like behaviors and hyperactivity of the lateral habenula (LHb), effects of dopamine (DA) D4 receptors in the LHb on depressive-like behaviors are unclear. Here we found that intra-LHb injection of the different doses of D4 receptor agonist A412997 and antagonist L741742 produced the different behavioral responses in SNc sham-lesioned rats, and only the high doses of A412997 and L741742 increased the expression of depressive-like behaviors or produced antidepressant-like effects in SNc-lesioned rats. The low doses of A412997 and L741742 altered the firing rate of LHb neurons and release of DA, GABA and glutamate in the LHb via the GABAergic rostromedial tegmental nucleus (RMTg) in SNc sham-lesioned rats, but not in SNc-lesioned rats. The high doses of A412997 and L741742 also altered the firing rate and release of the transmitters in both SNc sham-lesioned and SNc-lesioned rats, whereas these effects were not involved in the RMTg. Lesions of the SNc shortened the duration of significant effects on the firing rate and release of the transmitters induced by the high doses of A412997 and L741742. These findings suggest that D4 receptors in the LHb are involved in depression-like behaviors via the pre- and post-synaptic mechanisms and depletion of DA decreases the function and/or the expression of both pre- and post-synaptic D4 receptors. This study also points to the importance of the pre-synaptic D4 receptors in the regulation of Parkinson's disease-related depression.

Dopamine D4 receptor subtype activation reduces the rat cardiac parasympathetic discharge.

The dopaminergic system influences the heart rhythm by inhibiting the rat cardiac sympathetic and parasympathetic neurotransmissions through activation of D2-like receptors (encompassing the D2, D3, and D4 subtypes). Whereas D2 receptor subtype activation results in cardiac sympatho-inhibition, the dopamine receptor subtypes involved in rat cardiac vago-inhibition remain unknown. Hence, this study investigated the specific functional role of the D2-like receptor subtypes (D2, D3, and/or D4) inhibiting the rat heart cholinergic drive. For this purpose, male Wistar rats were pithed and prepared for cardiac vagal stimulation. Bradycardic responses were obtained by electrical stimulation of vagal fibres (3, 6, 9 Hz; n = 100) or i.v. acetylcholine (ACh; 1, 5, 10 µg/kg; n = 15). Expression of D2, D3, and D4 receptors was studied in left and right atrium samples by PCR (n = 4). Intravenous injections of quinpirole (D2-like agonist; 1-30 µg/kg), but not of SFK-38393 (D1-like agonist; 1-30 µg/kg), dose-dependently inhibited the vagally induced bradycardia. The vago-inhibition induced by quinpirole (which failed to affect the bradycardia to i.v. ACh) was unchanged after i.v. injections of the antagonists L-741,626 (D2; 100 µg/kg) or SB-277011-A (D3; 100 µg/kg), but it was abolished by L-745,870 (D4; 100 µg/kg). mRNA levels of D2, D3, and D4 receptor subtype were detected in the left and right rat atria. Our results suggest that the quinpirole-induced vagolytic effect involves prejunctional D4 receptor subtypes, located in the left and right atria. This provides new evidence on the relevance of D4 receptor modulating the heart parasympathetic control.

The effects of dopamine D4 receptor ligands on operant alcohol self-administration and cue- and stress-induced reinstatement in rats.

Dopamine, a neurotransmitter with 5 receptor subtypes, is critical to the dependence-forming properties of drugs of abuse. The role of the dopamine D4 receptor subtype in substance use disorders has remained somewhat elusive but the recent development of selective ligands holds promise for future investigations of this receptor subtype in substance use disorders, including alcohol use disorder. The purpose of the present study was to further elucidate the effects of a selective antagonist (L-745,870) and agonist (PD 168,077) on alcohol self-administration and reinstatement induced either by cues or stress. It was found that the D4 antagonist, but not agonist, reduced alcohol intake at the highest doses. Further, the D4 antagonist reduced stress-induced reinstatement, with no effects on cue-induced reinstatement; the agonist was without effect on either form of reinstatement. The dopamine D4 receptor antagonist was without effect on food reinforcement. This work deepens existing lines of evidence that the dopamine D4 receptor is involved in substance use disorders and suggests that dopamine D4 receptor blockade diminishes motivation for alcohol-taking without influencing natural food rewards. Furthermore, there appears to be a plausible effect of dopamine D4 receptor blockade interfering with stress- but not cue-induced alcohol-seeking.

Crystal structure of dopamine receptor D4 bound to the subtype selective ligand, L745870.

Multiple subtypes of dopamine receptors within the GPCR superfamily regulate neurological processes through various downstream signaling pathways. A crucial question about the dopamine receptor family is what structural features determine the subtype-selectivity of potential drugs. Here, we report the 3.5-angstrom crystal structure of mouse dopamine receptor D4 (DRD4) complexed with a subtype-selective antagonist, L745870. Our structure reveals a secondary binding pocket extended from the orthosteric ligand-binding pocket to a DRD4-specific crevice located between transmembrane helices 2 and 3. Additional mutagenesis studies suggest that the antagonist L745870 prevents DRD4 activation by blocking the relative movement between transmembrane helices 2 and 3. These results expand our knowledge of the molecular basis for the physiological functions of DRD4 and assist new drug design.

Design and synthesis of bridged piperidine and piperazine isosteres.

We have developed versatile methods toward the synthesis of a variety of piperidine/piperazine bridged isosteres of pridopidine. The compounds were assessed against the D2 receptor in agonist and antagonist modes and against the D4 receptor in agonist mode. hERG Binding and the ADME profiles were studied.

Bidirectional modulation of hippocampal synaptic plasticity by Dopaminergic D4-receptors in the CA1 area of hippocampus.

Long-term potentiation (LTP) is the persistent increase in the strength of the synapses. However, the neural networks would become saturated if there is only synaptic strenghthening. Synaptic weakening could be facilitated by active processes like long-term depression (LTD). Molecular mechanisms that facilitate the weakening of synapses and thereby stabilize the synapses are also important in learning and memory. Here we show that blockade of dopaminergic D4 receptors (D4R) promoted the formation of late-LTP and transformed early-LTP into late-LTP. This effect was dependent on protein synthesis, activation of NMDA-receptors and CaMKII. We also show that GABAA-receptor mediated mechanisms are involved in the enhancement of late-LTP. We could show that short-term plasticity and baseline synaptic transmission were unaffected by D4R inhibition. On the other hand, antagonizing D4R prevented both early and late forms of LTD, showing that activation of D4Rs triggered a dual function. Synaptic tagging experiments on LTD showed that D4Rs act as plasticity related proteins rather than the setting of synaptic tags. D4R activation by PD 168077 induced a slow-onset depression that was protein synthesis, NMDAR and CaMKII dependent. The D4 receptors, thus exert a bidirectional modulation of CA1 pyramidal neurons by restricting synaptic strengthening and facilitating synaptic weakening.

Return of D4 Dopamine Receptor Antagonists in Drug Discovery.

The dopamine D4 receptor garnered a great deal of interest in the early 1990s when studies showed the atypical antipsychotic clozapine possessed higher affinity for D4, relative to other dopamine receptor subtypes, and that this activity might underlie the unique clinical efficacy of clozapine. Unfortunately, D4 antagonists that were developed for schizophrenia failed in the clinic. Thus, D4 fell out of favor as a therapeutic target, and work in this area was silent for decades. Recently, D4 ligands with improved selectivity for D4 against not only D1-3,5 but also other biogenic amine targets have emerged, and D4 is once again in the spotlight as a novel target for both addiction and Parkinson's disease (PD), as well as other emerging diseases. This report will review the historical data for D4, review the known D4 ligands, and then highlight new data supporting a role for D4 inhibition in addiction, PD, and cancer.

Intravitreally-administered dopamine D2-like (and D4), but not D1-like, receptor agonists reduce form-deprivation myopia in tree shrews.

We examined the effect of intravitreal injections of D1-like and D2-like dopamine receptor agonists and antagonists and D4 receptor drugs on form-deprivation myopia (FDM) in tree shrews, mammals closely related to primates. In eleven groups (n = 7 per group), we measured the amount of FDM produced by monocular form deprivation (FD) over an 11-day treatment period. The untreated fellow eye served as a control. Animals also received daily 5 µL intravitreal injections in the FD eye. The reference group received 0.85% NaCl vehicle. Four groups received a higher, or lower, dose of a D1-like receptor agonist (SKF38393) or antagonist (SCH23390). Four groups received a higher, or lower, dose of a D2-like receptor agonist (quinpirole) or antagonist (spiperone). Two groups received the D4 receptor agonist (PD168077) or antagonist (PD168568). Refractions were measured daily; axial component dimensions were measured on day 1 (before treatment) and day 12. We found that in groups receiving the D1-like receptor agonist or antagonist, the development of FDM and altered ocular component dimensions did not differ from the NaCl group. Groups receiving the D2-like receptor agonist or antagonist at the higher dose developed significantly less FDM and had shorter vitreous chambers than the NaCl group. The D4 receptor agonist, but not the antagonist, was nearly as effective as the D2-like agonist in reducing FDM. Thus, using intravitreally-administered agents, we did not find evidence supporting a role for the D1-like receptor pathway in reducing FDM in tree shrews. The reduction of FDM by the dopamine D2-like agonist supported a role for the D2-like receptor pathway in the control of FDM. The reduction of FDM by the D4 receptor agonist, but not the D4 antagonist, suggests an important role for activation of the dopamine D4 receptor in the control of axial elongation and refractive development.

Activation of type 4 dopaminergic receptors in the prelimbic area of medial prefrontal cortex is necessary for the expression of innate fear behavior.

The prelimbic area (PL) of the medial Prefrontal cortex (mPFC) is involved in the acquisition and expression of conditioned and innate fear. Both types of fear share several neuronal pathways. It has been documented that dopamine (DA) plays an important role in the regulation of aversive memories in the mPFC. The exposure to an aversive stimulus, such as the smell of a predator odor or the exposure to footshock stress is accompanied by an increase in mPFC DA release. Evidence suggests that the type 4 dopaminergic receptor (D4R) is the molecular target through which DA modulates fear expression. In fact, the mPFC is the brain region with the highest expression of D4R; however, the role of D4R in the expression of innate fear has not been fully elucidated. Therefore, the principal objective of this work was to evaluate the participation of mPFC D4R in the expression of innate fear. Rats were exposed to the elevated plus-maze (EPM) and to the cat odor paradigm after the intra PL injection of L-745,870, selective D4R antagonist, to measure the expression of fear-related behaviors. Intra PL injection of L-745,870 increased the time spent in the EPM open arms and decreased freezing behavior in the cat odor paradigm. Our results also showed that D4R is expressed in GABAergic and pyramidal neurons in the PL region of PFC. Thus, D4R antagonism in the PL decreases the expression of innate fear-behavior indicating that the activation of D4R in the PL is necessary for the expression of innate fear-behavior.

Asenapine for the Control of Physical Aggression: A Prospective Naturalist Pilot Study.

It has been previously purported that higher relative affinity to the dopamine D4 receptor compared to D2 (i.e., D4/D2 affinity ratio > 1) may underlie unique antiaggression potency. Asenapine is a newer antipsychotic that also has D4/D2 affinity ratio > 1. It has demonstrated efficacy in reducing acute agitation in a placebo-controlled study. We performed a prospective naturalistic, pilot, proof of concept study on an inpatient psychiatric unit. Among patients with aggression at time of admission (≥ 12 on Refined Aggression Questionnaire [RAQ], or ≥ 2 on Modified Overt Aggression Scale [MOAS]), asenapine treatment was associated with a significant reduction in total aggression as measured by the MOAS (-14.7 ± 11.59 vs. -5.4 ± 10.12, P = 0.045), and particularly physical aggression (-8.0 ± 5.06 vs. -0.78 ± 2.40, P < 0.0001) compared to treatment that did not include asenapine. These data suggest that asenapine may be useful in the targeted treatment of aggression, and provide some support for the D4/D2 affinity ratio hypothesis.

The σ1 receptor regulates accumulation of GM1 ganglioside-enriched autophagosomes in astrocytes.

GM1 gangliosides (GM1) are acidic glycosphingolipids that are present in cell membranes and lipid raft domains, being particularly abundant in central nervous systems. GM1 participate in modulating cell membrane properties, intercellular recognition, cell regulation, and signaling. We previously demonstrated that GM1 are expressed inside astrocytes but not on the cell surface. We investigated whether the antipsychotic drug haloperidol induces GM1 expression in astrocytes, and found that the expression of GM1 was significantly upregulated by haloperidol in the intracellular vesicles of cultured astrocytes. The effects of haloperidol on GM1 expression acted through the σ1 receptor (σ1R), but not the dopamine-2 receptor. Inhibition of the ERK pathway blocked the induction of GM1 through the σ1R by haloperidol. Interestingly, this increase in GM1 expression induced the accumulation of autophagosomes in astrocytes. Moreover, the effect of haloperidol on the σ1R induced a decrease in GM1 in the cellular membrane of astrocytes. These findings suggested that the effects of haloperidol on the σ1R induced GM1 accumulation in the autophagosomes of astrocytes through activating the ERK pathway and a decrease in GM1 expression on the cell surface.

Discriminative stimulus properties of 1.25mg/kg clozapine in rats: Mediation by serotonin 5-HT2 and dopamine D4 receptors.

The atypical antipsychotic drug clozapine remains one of most effective treatments for schizophrenia, given a lack of extrapyramidal side effects, improvements in negative symptoms, cognitive impairment, and in symptoms in treatment-resistant schizophrenia. The adverse effects of clozapine, including agranulocytosis, make finding a safe clozapine-like a drug a goal for drug developers. The drug discrimination paradigm is a model of interoceptive stimulus that has been used in an effort to screen experimental drugs for clozapine-like atypical antipsychotic effects. The present study was conducted to elucidate the receptor-mediated stimulus properties that form this clozapine discriminative cue by testing selective receptor ligands in rats trained to discriminate a 1.25mg/kg dose of clozapine from vehicle in a two choice drug discrimination task. Full substitution occurred with the 5-HT2A inverse agonist M100907 and the two preferential D4/5-HT2/α1 receptor antagonists Lu 37-114 ((S)-1-(3-(2-(4-(1H-indol-5-yl)piperazin-1-yl)ethyl)indolin-1-yl)ethan-1-one) and Lu 37-254 (1-(3-(4-(1H-indol-5-yl)piperazin-1-yl)propyl)-3,4-dihydroquinolin-2(1H)-one). Partial substitution occurred with the D4 receptor antagonist Lu 38-012 and the α1 adrenoceptor antagonist prazosin. Drugs selective for 5-HT2C, 5-HT6 muscarinic, histamine H1, and benzodiazepine receptors did not substitute for clozapine. The present findings suggest that 5-HT2A inverse agonism and D4 receptor antagonism mediate the discriminative stimulus properties of 1.25mg/kg clozapine in rats, and further confirm that clozapine produces a complex compound discriminative stimulus.

Inhibition of Dopamine Receptor D4 Impedes Autophagic Flux, Proliferation, and Survival of Glioblastoma Stem Cells.

Glioblastomas (GBM) grow in a rich neurochemical milieu, but the impact of neurochemicals on GBM growth is largely unexplored. We interrogated 680 neurochemical compounds in patient-derived GBM neural stem cells (GNS) to determine the effects on proliferation and survival. Compounds that modulate dopaminergic, serotonergic, and cholinergic signaling pathways selectively affected GNS growth. In particular, dopamine receptor D4 (DRD4) antagonists selectively inhibited GNS growth and promoted differentiation of normal neural stem cells. DRD4 antagonists inhibited the downstream effectors PDGFRß, ERK1/2, and mTOR and disrupted the autophagy-lysosomal pathway, leading to accumulation of autophagic vacuoles followed by G0/G1 arrest and apoptosis. These results demonstrate a role for neurochemical pathways in governing GBM stem cell proliferation and suggest therapeutic approaches for GBM.

Synthesis and characterization of a series of chiral alkoxymethyl morpholine analogs as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a series of chiral alkoxymethyl morpholine analogs. Our efforts have culminated in the identification of (S)-2-(((6-chloropyridin-2-yl)oxy)methyl)-4-((6-fluoro-1H-indol-3-yl)methyl)morpholine as a novel potent and selective dopamine D4 receptor antagonist with selectivity against the other dopamine receptors tested (<10% inhibition at 1µM against D1, D2L, D2S, D3, and D5).

Dopamine-dependent synaptic plasticity in an amygdala inhibitory circuit controls fear memory expression.

Of the numerous events that occur in daily life, we readily remember salient information, but do not retain most less-salient events for a prolonged period. Although some of the episodes contain putatively emotional aspects, the information with lower saliency is rarely stored in neural circuits via an unknown mechanism. We provided substantial evidence indicating that synaptic plasticity in the dorsal ITC of amygdala allows for selective storage of salient emotional experiences, while it deters less-salient experience from entering long-term memory. After activation of D4R or weak fear conditioning, STDP stimulation induces LTD in the LA-ITC synapses. This form of LTD is dependent upon presynaptic D4R, and is likely to result from enhancement of GABA release. Both optogenetic abrogation of LTD and ablation of D4R at the dorsal ITC in vivo lead to heightened and over-generalized fear responses. Finally, we demonstrated that LTD was impaired at the dorsal ITC of PTSD model mice, which suggests that maladaptation of GABAergic signaling and the resultant LTD impairment contribute to the endophenotypes of PTSD.