D3 Receptor Ligands Research Articles

Aromatic linker variations in novel dopamine D2 and D3 receptor ligands.

Dopamine D2-like receptors, especially D2 and D3 receptor subtypes, are important targets of antipsychotic agents. Many of these antipsychotics share an aliphatic linker element between a protonable amine group and an acyl-like moiety. Here, we have modified this aliphatic linker into phenylmethyl and phenylethyl linkers substituted in different positions. The design, synthesis, and in vitro evaluation of 18 dopamine D2 and D3 receptor ligands were performed in this study. Using a radioligand displacement assay, all ligands were found to have modest nanomolar affinity to D2R and D3R. N-(4-{2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl}phenyl)acetamide (6c) demonstrates the highest D3R and D2R affinity values (pKi values of 7.83 [D2R] and 8.04 [D3R]), featuring a slight preference to D3R. This derivative can be taken as a reference structure for the development of a new class of D2R and D3R ligands.

In vitro characterization of [125I]HY-3-24, a selective ligand for the dopamine D3 receptor.

Dopamine D3 receptor (D3R) ligands have been studied for the possible treatment of neurological and neuropsychiatric disorders. However, selective D3R radioligands for in vitro binding studies have been challenging to identify due to the high structural similarity between the D2R and D3R. In a prior study, we reported a new conformationally-flexible benzamide scaffold having a high affinity for D3R and excellent selectivity vs. D2R. In the current study, we characterized the in vitro binding properties of a new radioiodinated ligand, [125I]HY-3-24. In vitro binding studies were conducted in cell lines expressing D3 receptors, rat striatal homogenates, and rat and non-human primate (NHP) brain tissues to measure regional brain distribution of this radioligand. HY-3-24 showed high potency at D3R (Ki = 0.67 ± 0.11 nM, IC50 = 1.5 ± 0.58 nM) compared to other D2-like dopamine receptor subtypes (D2R Ki = 86.7 ± 11.9 nM and D4R Ki > 1,000). The Kd (0.34 ± 0.22 nM) and Bmax (38.91 ± 2.39 fmol/mg) values of [125I]HY-3-24 were determined. In vitro binding studies in rat striatal homogenates using selective D2R and D3R antagonists confirmed the D3R selectivity of [125I]HY-3-24. Autoradiography results demonstrated that [125I]HY-3-24 specifically binds to D3Rs in the nucleus accumbens, islands of Calleja, and caudate putamen in rat and NHP brain sections. These results suggest that [125I]HY-3-24 appears to be a novel radioligand that exhibits high affinity binding at D3R, with low binding to other D2-like dopamine receptors. It is anticipated that [125I]HY-3-24 can be used as the specific D3R radioligand.

NeuroClick: software for mimicking click reaction to generate drug-like molecules permeating the blood-brain barrier.

Background: Traditional methods for chemical library generation in virtual screening often impose limitations on the accessible chemical space or produce synthetically irrelevant structures. Incorporating common chemical reactions into generative algorithms could offer significant benefits. Materials & methods: In this study, we developed NeuroClick, a graphical user interface software designed to perform in silico azide-alkyne cycloaddition, a widely utilized synthetic approach in modern medicinal chemistry. Results & conclusion: NeuroClick facilitates the generation and filtering of large combinatorial libraries at a remarkable rate of 10,000 molecules per minute. Moreover, the generated products can be filtered to identify subsets of pharmaceutically relevant compounds based on Lipinski's rule of five and blood-brain barrier permeability prediction. We demonstrate the utility of NeuroClick by generating and filtering several thousand molecules for dopamine D3 receptor ligand screening.

Humanized dopamine D4.7 receptor male mice display risk-taking behavior and deficits of social recognition and working memory in light/dark-dependent manner.

The dopamine D4 receptor 7-repeat allele (D4.7 R) has been linked with psychiatric disorders such as attention-deficit-hyperactivity disorder, autism, and schizophrenia. However, the highly diverse study populations and often contradictory findings make it difficult to draw reliable conclusions. The D4.7 R has the potential to explain individual differences in behavior. However, there is still a great deal of ambiguity surrounding whether it is causally connected to the etiology of psychiatric disorders. Therefore, humanized D4.7 R mice, with the long third intracellular domain of the human D4.7 R, may provide a valuable tool to examine the relationship between the D4.7 R variant and specific behavioral phenotypes. We report that D4.7 R male mice carrying the humanized D4.7 R variant exhibit distinct behavioral features that are dependent on the light-dark cycle. The behavioral phenotype was characterized by a working memory deficit, delayed decision execution in the light phase, decreased stress and anxiety, and increased risk behavior in the dark phase. Further, D4.7 R mice displayed impaired social recognition memory in both the light and dark phases. These findings provide insight into the potential causal relationship between the human D4.7 R variant and specific behaviors and encourage further consideration of dopamine D4 receptor (DRD4) ligands as novel treatments for psychiatric disorders in which D4.7 R has been implicated.

Design and synthesis of fused azacyclic and azaspirocyclic γ-lactams targeting dopaminergic D2/D4 receptors

The synthesis of fused azabicyclic and azaspirobicyclic γ-lactams corresponding to tetrahydro-1H-pyrrolo[3,4-c]pyrrol-4-one and 2,7-diazaspiro[4.4]nonane-1-one core structures respectively containing a variety of N-aryl substituents is described as potential agonists of the dopaminergic D2/D4 receptors. Using the risperidone/DRD2 crystal structure data, we performed docking studies on a prototypical synthetic fused azabicyclic γ-lactam, and the known D2 receptor ligand 1-(2-methoxyphenyl)-4-propylpiperazine. The racemic or enantiopure tosylate salts in the fused azabicyclic γ-lactam series were found to be devoid of activity ((≤ 10 % inhibition for either receptor at 0.1 µM). However, marginal activity was expressed by the azaspirobicyclic γ-lactam series against the D4 receptor albeit at 10 µM with favorable ADME properties. The fused and spiroazabicyclic γ-lactam core structures are interesting ring-constrained piperazines for further diversification in the context of medicinally relevant programs.

Pharmacological and Physicochemical Properties Optimization for Dual-Target Dopamine D3 (D3R) and μ-Opioid (MOR) Receptor Ligands as Potentially Safer Analgesics.

A new generation of dual-target μ opioid receptor (MOR) agonist/dopamine D3 receptor (D3R) antagonist/partial agonists with optimized physicochemical properties was designed and synthesized. Combining in vitro cell-based on-target/off-target affinity screening, in silico computer-aided drug design, and BRET functional assays, we identified new structural scaffolds that achieved high affinity and agonist/antagonist potencies for MOR and D3R, respectively, improving the dopamine receptor subtype selectivity (e.g., D3R over D2R) and significantly enhancing central nervous system multiparameter optimization scores for predicted blood-brain barrier permeability. We identified the substituted trans-(2S,4R)-pyrrolidine and trans-phenylcyclopropyl amine as key dopaminergic moieties and tethered these to different opioid scaffolds, derived from the MOR agonists TRV130 (3) or loperamide (6). The lead compounds 46, 84, 114, and 121 have the potential of producing analgesic effects through MOR partial agonism with reduced opioid-misuse liability via D3R antagonism. Moreover, the peripherally limited derivatives could have therapeutic indications for inflammation and neuropathic pain.

Exploring Structural Determinants of Bias among D4 Subtype-Selective Dopamine Receptor Agonists.

The high affinity dopamine D4 receptor ligand APH199 and derivatives thereof exhibit bias toward the Gi signaling pathway over β-arrestin recruitment compared to quinpirole. Based on APH199, two novel groups of D4 subtype selective ligands were designed and evaluated, in which the original benzyl phenylsemicarbazide substructure was replaced by either a biphenylmethyl urea or a biphenyl urea moiety. Functional assays revealed a range of different bias profiles among the newly synthesized compounds, namely, with regard to efficacy, potency, and GRK2 dependency, in which bias factors range from 1 to over 300 and activation from 15% to over 98% compared to quinpirole. These observations demonstrate that within bias, an even more precise tuning toward a particular profile is possible, which─in a general sense─could become an important aspect in future drug development. Docking studies enabled further insight into the role of the ECL2 and the EPB in the emergence of bias, thereby taking advantage of the diversity of functionally selective D4 agonists now available.

Impact of membrane lipid polyunsaturation on dopamine D2 receptor ligand binding and signaling.

Increasing evidence supports a relationship between lipid metabolism and mental health. In particular, the biostatus of polyunsaturated fatty acids (PUFAs) correlates with some symptoms of psychiatric disorders, as well as the efficacy of pharmacological treatments. Recent findings highlight a direct association between brain PUFA levels and dopamine transmission, a major neuromodulatory system implicated in the etiology of psychiatric symptoms. However, the mechanisms underlying this relationship are still unknown. Here we demonstrate that membrane enrichment in the n-3 PUFA docosahexaenoic acid (DHA), potentiates ligand binding to the dopamine D2 receptor (D2R), suggesting that DHA acts as an allosteric modulator of this receptor. Molecular dynamics simulations confirm that DHA has a high preference for interaction with the D2R and show that membrane unsaturation selectively enhances the conformational dynamics of the receptor around its second intracellular loop. We find that membrane unsaturation spares G protein activity but potentiates the recruitment of β-arrestin in cells. Furthermore, in vivo n-3 PUFA deficiency blunts the behavioral effects of two D2R ligands, quinpirole and aripiprazole. These results highlight the importance of membrane unsaturation for D2R activity and provide a putative mechanism for the ability of PUFAs to enhance antipsychotic efficacy.

Identifying Dopamine D3 Receptor Ligands through Virtual Screening and Exploring the Binding Modes of Hit Compounds.

The dopamine D3 receptor (D3R) is an important central nervous system target for treating various neurological diseases. D3R antagonists modulate the improvement of psychostimulant addiction and relapse, while D3R agonists can enhance the response to dopaminergic stimulation and have potential applications in treating Parkinson's disease, which highlights the importance of identifying novel D3R ligands. Therefore, we performed auto dock Vina-based virtual screening and D3R-binding-affinity assays to identify human D3R ligands with diverse structures. All molecules in the ChemDiv library (>1,500,000) were narrowed down to a final set of 37 molecules for the binding assays. Twenty-seven compounds exhibited over 50% inhibition of D3R at a concentration of 10 μM, and 23 compounds exhibited over 70% D3R inhibition at a concentration of 10 μM. Thirteen compounds exhibited over 80% inhibition of D3R at a concentration of 10 μM and the IC50 values were measured. The IC50 values of the five compounds with the highest D3R-inhibition rates ranged from 0.97 μM to 1.49 μM. These hit compounds exhibited good structural diversity, which prompted us to investigate their D3R-binding modes. After trial and error, we combined unbiased molecular dynamics simulation (MD) and molecular mechanics generalized Born surface area (MM/GBSA) binding free-energy calculations with the reported protein-ligand-binding pose prediction method using induced-fit docking (IFD) and binding pose metadynamics (BPMD) simulations into a self-consistent and computationally efficient method for predicting and verifying the binding poses of the hit ligands to D3R. Using this IFD-BPMD-MD-MM/GBSA method, we obtained more accurate and reliable D3R-ligand-binding poses than were obtained using the reported IFD-BPMD method. This IFD-BPMD-MD-MM/GBSA method provides a novel paradigm and reference for predicting and validating other protein-ligand binding poses.

Phosphoproteomic Analysis of Dopamine D2 Receptor Signaling Reveals Interplay of G Protein- and β-Arrestin-Mediated Effects.

Leveraging biased signaling of G protein-coupled receptors has been proposed as a promising strategy for the development of drugs with higher specificity. However, the consequences of selectively targeting G protein- or β-arrestin-mediated signaling on cellular functions are not comprehensively understood. In this study, we utilized phosphoproteomics to gain a systematic overview of signaling induced by the four biased and balanced dopamine D2 receptor (D2R) ligands MS308, BM138, quinpirole, and sulpiride in an in vitro D2R transfection model. Quantification of 14,160 phosphosites revealed a low impact of the partial G protein agonist MS308 on cellular protein phosphorylation, as well as surprising similarities between the balanced agonist quinpirole and the inverse agonist sulpiride. Analysis of the temporal profiles of ligand-induced phosphorylation events showed a transient impact of the G protein-selective agonist MS308, whereas the β-arrestin-preferring agonist BM138 elicited a delayed, but more pronounced response. Functional enrichment analysis of ligand-impacted phosphoproteins and treatment-linked kinases confirmed multiple known functions of D2R signaling while also revealing novel effects, for example of MS308 on sterol regulatory element-binding protein-related gene expression. All raw data were deposited in MassIVE (MSV000089457).

In vivo and in vitro binding of [125 I]I-R-(+)-TISCH: A dopamine D1 receptor ligand for studying pancreatic β-cell mass.

Diabetes mellitus (DM) and insulinoma are mainly affected by the status of pancreatic β-cell mass (BCM). Development of imaging agents for BCM allows to study pancreatic β cells and the relationship between β cells and DM or insulinoma. In this study, we investigated the density of dopamine D1 receptor on the β cells and measured BCM by statistical image processing. The pancreatic uptakes of [125 I]I-R-(+)-7-chloro-8-hydroxy-1-(3'-iodopheny1)-3-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([125 I]I-R-(+)-TISCH), dopamine D1 receptor tracer, in normal and diabetic rats displayed significant differences at 30 min (1.11 ± 0.08% ID/g vs. 0.63 ± 0.09% ID/g, p < 0.0001). In the presence of SCH23390, the pancreatic uptake of [125 I]I-R-(+)-TISCH at 30 min in normal rats was lower (1.01 ± 0.04% ID/g, p < 0.05). Although the blocking was not complete, [125 I]I-R-(+)-TISCH showed specific binding signals to the pancreas. Furthermore, the uptakes of [125 I]I-R-(+)-TISCH in INS-1 cells were reduced in the presence of SCH23390 at different concentrations. [125 I]I-R-(+)-TISCH displayed a respectable uptake in insulinoma. Overall, [125 I]I-R-(+)-TISCH provided specific binding signals to pancreatic β cells. Although the specific signal may not be sufficient for imaging in vivo, the dopamine D1 receptor can still be considered as a potential target for studying BCM. Further investigation will be required to optimize the ligand.

Recent advances in dopamine D2 receptor ligands in the treatment of neuropsychiatric disorders.

Dopamine is a biologically active amine synthesized in the central and peripheral nervous system. This biogenic monoamine acts by activating five types of dopamine receptors (D1-5 Rs), which belong to the G protein-coupled receptor family. Antagonists and partial agonists of D2 Rs are used to treat schizophrenia, Parkinson's disease, depression, and anxiety. The typical pharmacophore with high D2 R affinity comprises four main areas, namely aromatic moiety, cyclic amine, central linker and aromatic/heteroaromatic lipophilic fragment. From the literature reviewed herein, we can conclude that 4-(2,3-dichlorophenyl), 4-(2-methoxyphenyl)-, 4-(benzo[b]thiophen-4-yl)-1-substituted piperazine, and 4-(6-fluorobenzo[d]isoxazol-3-yl)piperidine moieties are critical for high D2 R affinity. Four to six atoms chains are optimal for D2 R affinity with 4-butoxyl as the most pronounced one. The bicyclic aromatic/heteroaromatic systems are most frequently occurring as lipophilic appendages to retain high D2 R affinity. In this review, we provide a thorough overview of the therapeutic potential of D2 R modulators in the treatment of the aforementioned disorders. In addition, this review summarizes current knowledge about these diseases, with a focus on the dopaminergic pathway underlying these pathologies. Major attention is paid to the structure, function, and pharmacology of novel D2 R ligands, which have been developed in the last decade (2010-2021), and belong to the 1,4-disubstituted aromatic cyclic amine group. Due to the abundance of data, allosteric D2 R ligands and D2 R modulators from patents are not discussed in this review.

Identification of Novel Dopamine D2 Receptor Ligands-A Combined In Silico/In Vitro Approach.

Diseases of the central nervous system are an alarming global problem showing an increasing prevalence. Dopamine receptor D2 (D2R) has been shown to be involved in central nervous system diseases. While different D2R-targeting drugs have been approved by the FDA, they all suffer from major drawbacks due to promiscuous receptor activity leading to adverse effects. Increasing the number of potential D2R-targeting drug candidates bears the possibility of discovering molecules with less severe side-effect profiles. In dire need of novel D2R ligands for drug development, combined in silico/in vitro approaches have been shown to be efficient strategies. In this study, in silico pharmacophore models were generated utilizing both ligand- and structure-based approaches. Subsequently, different databases were screened for novel D2R ligands. Selected virtual hits were investigated in vitro, quantifying their binding affinity towards D2R. This workflow successfully identified six novel D2R ligands exerting micro- to nanomolar (most active compound KI = 4.1 nM) activities. Thus, the four pharmacophore models showed prospective true-positive hit rates in between 4.5% and 12%. The developed workflow and identified ligands could aid in developing novel drug candidates for D2R-associated pathologies.

Receptor density influences the recruitment bias of aripiprazole and brexpiprazole at the dopamine D2L receptor.

Aripiprazole, brexpiprazole, and cariprazine are dopamine D2 receptor ligands considered as effective and tolerable antipsychotics. Brain imaging studies showed that schizophrenia is characterized by elevated dopamine receptor density, which is exacerbated by antipsychotic treatments. Despite the complexity of translating in vitro studies to human neurobiology, overexpression experiments in transfected cells provide a proof-of-concept model of the influence of receptor density on antipsychotic treatments. Since receptor density was demonstrated to influence the signaling profile of dopaminergic ligands, we hypothesized that high dopamine D2 receptor expression levels could influence the recruitment of Gi1 and β-arrestin2 in response to partial agonists used as antipsychotics. A nanoluciferase complementation assay was used to monitor β-arrestin2 and Gi1 recruitment at the dopamine D2L receptor in response to aripiprazole, brexpiprazole, and cariprazine. This was performed in transfected cells carrying a doxycycline-inducible system allowing to manipulate the expression of the dopamine D2L receptors. Increasing D2L receptor density reoriented aripiprazole's preferential recruitment from Gi1 to β-arrestin2. With respect to brexpiprazole, which showed inverse agonism for β-arrestin2 recruitment at the lower receptor density tested, inverse agonism for Gi1 recruitment was observed when tested at a high receptor expression level. At variance, cariprazine evoked a potent partial agonism for β-arrestin2 recruitment only, in all the tested conditions. D2L receptor density appears to shape the recruitment bias of aripiprazole and brexpiprazole, but not cariprazine. This suggests that changes in receptor expression level could qualitatively influence the functional response of partial agonists used in psychiatry.

Further studies on C2′-substituted 1-phenylbenzazepines as dopamine D1 receptor ligands

The 1-phenylbenzazepine scaffold has yielded several D1R targeting ligands, but some gaps remain in our understanding of the structure–activity relationships in this scaffold. In particular, there is a paucity of studies that have investigated the effects of substituents at the C2′ position of 1-phenylbenzazepines on their affinity and selectivity towards D1R. In this study, a set of methyl- and fluoro- C2′-substituted 1-phenylbenzazepines, with ring A catechol or 8-hydroxy-7-methoxy moieties in tandem with N-methyl or N-allyl substituent groups, was synthesized and evaluated for affinity at a subset of dopamine receptors - D1R, D2R and D5R. These studies indicate that an N-methyl group is generally preferred over N-unsubstituted or N-allyl groups for strong D1R affinity. In addition, it was revealed that compounds with a ring A 8-hydroxy-7-methoxy motif displayed stronger D1R affinity than analogous compounds with a ring A catechol moiety. Furthermore, the presence of a C2′ substituent does not significantly impact D1R selectivity over D5R. However, for all analogs assessed, D1R selectivity over D2R was maintained. D1R vs D5R selectivity was generally poor or modest (less than 10-fold) among members of the series. A new high affinity selective D1R ligand − 10b (Ki = 5.7 nM), was identified in this study; further pharmacological characterization indicates that 10b is an antagonist at D1R (IC50 = 10.7 nM). Docking studies on 10b indicate that a number of interactions with hydrophobic residues (Trp321, Val317, Phe313, Phe289, Phe288, Phe285, Phe203, Tyr194, Leu190, Ser188, His 164, Ile104, Val100 and Trp99) in addition to the typical N-Asp103 salt bridge are important for its D1R affinity.

Investigating the ligand agonism and antagonism at the D2long receptor by dynamic mass redistribution

The signalling of the D2 receptor (D2R), a G protein-coupled receptor (GPCR), is a complex process consisting of various components. For the screening of D2R ligands, methods quantifying distinct second messengers such as cAMP or the interaction of the receptor with β-arrestin, are commonly employed. In contrast, a label-free biosensor technology like dynamic mass redistribution (DMR), where it is mostly unknown how the individual signalling pathways contribute to the DMR signal, provides a holistic readout of the complex cellular response. In this study, we report the successful application of the DMR technology to CHO-K1 cells stably expressing the human dopamine D2long receptor. In real-time kinetic experiments, studies of D2R reference compounds yielded results for agonists and antagonists that were consistent with those obtained by conventional methods and also allowed a discrimination between partial and full agonists. Furthermore, investigations on the signalling pathway in CHO-K1 hD2longR cells identified the Gαi/o protein as the main proximal trigger of the observed DMR response. The present study has shown that the DMR technology is a valuable method for the characterisation of putative new ligands and, due to its label-free nature, suggests its use for deorphanisation studies of GPCRs.

Indirect SPECT Imaging Evaluation for Possible Nose-to-Brain Drug Delivery Using a Compound with Poor Blood-Brain Barrier Permeability in Mice.

Single-photon emission computed tomography (SPECT) imaging using intravenous radioactive ligand administration to indirectly evaluate the time-dependent effect of intranasal drugs with poor blood-brain barrier permeability on brain drug distributions in mice was evaluated. The biodistribution was examined using domperidone, a dopamine D2 receptor ligand, as the model drug, with intranasal administration at 0, 15, or 30 min before intravenous [123I]IBZM administration. In the striatum, [123I]IBZM accumulation was significantly lower after intranasal (IN) domperidone administration than in controls 15 min after intravenous [125I]IBZM administration. [123I]IBZM SPECT was acquired with intravenous (IV) or IN domperidone administration 15 min before [123I]IBZM, and time–activity curves were obtained. In the striatum, [123I]IBZM accumulation was clearly lower in the IN group than in the control and IV groups. Time–activity curves showed no significant difference between the control and IV groups in the striatum, and values were significantly lowest during the first 10 min in the IN group. In the IN group, binding potential and % of receptor occupancy were significantly lower and higher, respectively, compared to the control and IV groups. Thus, brain-migrated domperidone inhibited D2R binding of [123I]IBZM. SPECT imaging is suitable for research to indirectly explore nose-to-brain drug delivery and locus-specific biological distribution.

Design, synthesis, and evaluation of functionalized 5-(4-arylpiperazin-1-yl)-N-quinolinyl-pentanamides as selective dopamine D3 receptor ligands

Design, synthesis, and evaluation of functionalized 5-(4-arylpiperazin-1-yl)-N-quinolinyl-pentanamides as selective dopamine D3 receptor ligands

Synthesis of functionalized 5-(4-arylpiperazin-1-yl)-N-arylpentanamides and their evaluation as D3 receptor ligands

Synthesis of functionalized 5-(4-arylpiperazin-1-yl)-N-arylpentanamides and their evaluation as D3 receptor ligands

Synthesis and evaluation of cyclic diamino benzamide based D3 receptor ligands

Dopamine (1) is a key neurotransmitter whose impact on pharmacological processes is mediated by a family of dopamine receptors designated D1, D2, D3, D4, and D5. Various diseases and conditions such as schizophrenia, drug abuse, depression, restless leg syndrome, Parkinson’s disease (PD), and inflammatory diseases have been linked to aberrant D3 activity. Herein, we report a series of novel D3 ligands with improved solubility over our previous lead compound, MC25-41 (2).