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 ß-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.

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.

Benzyl Phenylsemicarbazides: A Chemistry-Driven Approach Leading to G Protein-Biased Dopamine D4 Receptor Agonists with High Subtype Selectivity.

Many subtype-selective dopamine receptor ligands developed for the D2-D4 family incorporate a 1-arylpiperazine-derived primary recognition motif, which is connected to a lipophilic moiety occupying an extended binding pocket (EBP) of the receptor via an aliphatic linker of variable lengths. The evaluation of a novel group of dopamine receptor ligands now showed that highly subtype-selective ligands [up to Ki(D4.4) = 0.25 nM, D2L/D4.4 = 320, D3/D4.4 = 710 for APH199 (17)] can be obtained by choosing a relatively large and conformationally flexible 1-benzyl-1-phenylsemicarbazide substructure to fill the EBP. The novel chemotype APH199 (17) was found to act as a full agonist at the D4 receptor showing significant bias toward G protein activation over ß-arrestin recruitment in comparison to quinpirole.

Ultra-large library docking for discovering new chemotypes.

Despite intense interest in expanding chemical space, libraries containing hundreds-of-millions to billions of diverse molecules have remained inaccessible. Here we investigate structure-based docking of 170 million make-on-demand compounds from 130 well-characterized reactions. The resulting library is diverse, representing over 10.7 million scaffolds that are otherwise unavailable. For each compound in the library, docking against AmpC ß-lactamase (AmpC) and the D4 dopamine receptor were simulated. From the top-ranking molecules, 44 and 549 compounds were synthesized and tested for interactions with AmpC and the D4 dopamine receptor, respectively. We found a phenolate inhibitor of AmpC, which revealed a group of inhibitors without known precedent. This molecule was optimized to 77 nM, which places it among the most potent non-covalent AmpC inhibitors known. Crystal structures of this and other AmpC inhibitors confirmed the docking predictions. Against the D4 dopamine receptor, hit rates fell almost monotonically with docking score, and a hit-rate versus score curve predicted that the library contained 453,000 ligands for the D4 dopamine receptor. Of 81 new chemotypes discovered, 30 showed submicromolar activity, including a 180-pM subtype-selective agonist of the D4 dopamine receptor.

1-[3-(4-Butylpiperidin-1-yl)propyl]-1,2,3,4-tetrahydroquinolin-2-one (77-LH-28-1) as a Model for the Rational Design of a Novel Class of Brain Penetrant Ligands with High Affinity and Selectivity for Dopamine D4 Receptor.

In the present article, the M1 mAChR bitopic agonist 1-[3-(4-butylpiperidin-1-yl)propyl]-1,2,3,4-tetrahydroquinolin-2-one (77-LH-28-1, 1) has been demonstrated to show unexpected D4R selectivity over D2R and D3R and to behave as a D4R antagonist. To better understand the structural features required for the selective interaction with the D4R and to obtain compounds unable to activate mAChRs, the aliphatic butyl chain and the piperidine nucleus of 1 were modified, affording compounds 2-14. The 4-benzylpiperidine 9 and the 4-phenylpiperazine 12 showed high D4R affinity and selectivity not only over the other D2-like subtypes, but also over M1-M5 mAChRs. Derivative 12 was also highly selective over some selected off-targets. This compound showed biased behavior, potently and partially activating Gi protein and inhibiting ß-arrestin2 recruitment in functional studies. Pharmacokinetic studies demonstrated that it was characterized by a relevant brain penetration. Therefore, 12 might be a useful tool to better clarify the role played by D4R in disorders in which this subtype is involved.

Structure of the D2 dopamine receptor bound to the atypical antipsychotic drug risperidone.

Dopamine is a neurotransmitter that has been implicated in processes as diverse as reward, addiction, control of coordinated movement, metabolism and hormonal secretion. Correspondingly, dysregulation of the dopaminergic system has been implicated in diseases such as schizophrenia, Parkinson's disease, depression, attention deficit hyperactivity disorder, and nausea and vomiting. The actions of dopamine are mediated by a family of five G-protein-coupled receptors. The D2 dopamine receptor (DRD2) is the primary target for both typical and atypical antipsychotic drugs, and for drugs used to treat Parkinson's disease. Unfortunately, many drugs that target DRD2 cause serious and potentially life-threatening side effects due to promiscuous activities against related receptors. Accordingly, a molecular understanding of the structure and function of DRD2 could provide a template for the design of safer and more effective medications. Here we report the crystal structure of DRD2 in complex with the widely prescribed atypical antipsychotic drug risperidone. The DRD2-risperidone structure reveals an unexpected mode of antipsychotic drug binding to dopamine receptors, and highlights structural determinants that are essential for the actions of risperidone and related drugs at DRD2.

Isopeptide and ester bond ubiquitination both regulate degradation of the human dopamine receptor 4.

How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R.

D4 dopamine receptor high-resolution structures enable the discovery of selective agonists.

Dopamine receptors are implicated in the pathogenesis and treatment of nearly every neuropsychiatric disorder. Although thousands of drugs interact with these receptors, our molecular understanding of dopaminergic drug selectivity and design remains clouded. To illuminate dopamine receptor structure, function, and ligand recognition, we determined crystal structures of the D4 dopamine receptor in its inactive state bound to the antipsychotic drug nemonapride, with resolutions up to 1.95 angstroms. These structures suggest a mechanism for the control of constitutive signaling, and their unusually high resolution enabled a structure-based campaign for new agonists of the D4 dopamine receptor. The ability to efficiently exploit structure for specific probe discovery-rapidly moving from elucidating receptor structure to discovering previously unrecognized, selective agonists-testifies to the power of structure-based approaches.

Palmitoylation of the carboxyl-terminal tail of dopamine D4 receptor is required for surface expression, endocytosis, and signaling.

The amino acid sequences and signaling pathways of D2-like dopamine receptors (D2R, D3R, and D4R) are highly conserved. D4R has been suggested to be associated with novelty seeking, and binds with high affinity to an atypical antipsychotic drug with fewer motor function-related side effects than typical neuroleptics. A study comparing D2R and D3R reported that palmitoylation is important for the proper functioning of D3R. Although D4R is a member of the D2-like receptor family, its palmitoylation status and the functional significance of any such posttranslational modification are unknown. In this study, it was found that D4-4, an alternatively spliced form of D4R, was palmitoylated on Cys467, the terminal amino acid residue of the receptor. When palmitoylation of D4R was inhibited, by either mutation of the consensus site or by treatment with the palmitoylation inhibitor 2-bromopalmitate (2BP), D4R cell surface expression, signaling, and endocytosis were all impaired. Exchanging the carboxyl-terminal tails of D2R and D4R resulted in a switching of the palmitoylation phenotype, as well as concomitant changes in functionality, such as receptor surface expression, endocytosis, and signaling. Despite the high degree of homology in the carboxyl-terminal tails of D2-like receptors, palmitoylation occurs exclusively in the D3R and D4R family members, with the D4R tail being sufficient to endow D2R with palmitoylation-associated functionality. Thus, this study provides new insights into a consensus sequence for palmitoylation, as well as possible strategies for the regulation of D4R, which has implications for the treatment of various neurological and psychiatric disorders.

Structural Insights into 5-HT1A/D4 Selectivity of WAY-100635 Analogues: Molecular Modeling, Synthesis, and in Vitro Binding.

The resurgence of interest in 5-HT1A receptors as a therapeutic target requires the existence of highly selective 5-HT1A ligands. To date, WAY-100635 has been the prototypical antagonist of these receptors. However, this compound also has significant affinity for and activity at D4 dopamine receptors. In this context, this work was aimed at better understanding the 5-HT1A/D4 selectivity of WAY-100635 and analogues from a structural point of view. In silico investigations revealed two key interactions for the 5-HT1A/D4 selectivity of WAY-100635 and analogues. First, a hydrogen bond only found with the Ser 7.36 of D4 receptor appeared to be the key for a higher D4 affinity for newly synthesized aza analogues. The role of Ser 7.36 was confirmed as the affinity of aza analogues for the mutant D4 receptor S7.36A was reduced. Then, the formation of another hydrogen bond with the conserved Ser 5.42 residue appeared to be also critical for D4 binding.

Dopamine D4 receptor ubiquitination.

Ubiquitination is a post-translational modification that targets proteins for degradation but can also regulate other cellular processes such as endocytosis, trafficking and DNA repair. We investigate ubiquitination of the dopamine D4receptor (D4R) which belongs to the superfamily of G protein-coupled receptors (GPCR). Several polymorphic variants of the D4R exist, which differ in the number of 16-amino acid repeats in the third intracellular loop (IC3) of the receptor. The functional role of this polymorphic region is not known but persons with the seven-repeat allele show a predisposition to develop attention deficit hyperactivity disorder (ADHD). We identified a protein, KLHL12, which specifically interacts with this polymorphic region and enhances ubiquitination of the D4R. We have tested the influence of KLHL12 on the ubiquitination of the most common D4R polymorphic variants and found that KLHL12 strongly promotes ubiquitination of the two- and four-repeat variant but has hardly any effect on ubiquitination of the seven-repeat D4R. This suggests that differential ubiquitination of the D4R may have functional implications. Moreover, we were able to demonstrate that KLHL12-mediated D4R ubiquitination does not lead to receptor degradation. Next, we aimed to identify specific residues in the sequence of D4R which undergo ubiquitination and observed that the lysine-less receptor mutant is still ubiquitinated. Subsequently, we have tested the hypothesis whether KLHL12 could promote ubiquitination on non-lysine residues of the D4R. The importance of the cysteine and serine/threonine residues in the ubiquitination process of the receptor was examined and the obtained results confirmed that D4R can be ubiquitinated on non-lysine residues. In this review we summarize our data on D4R ubiquitination and put this in the light of other GPCR ubiquitination studies.

Structural signatures of DRD4 mutants revealed using molecular dynamics simulations: Implications for drug targeting.

Human Dopamine Receptor D4 (DRD4) orchestrates several neurological functions and represents a target for many psychological disorders. Here, we examined two rare variants in DRD4; V194G and R237L, which elicit functional alterations leading to disruption of ligand binding and G protein coupling, respectively. Using atomistic molecular dynamics (MD) simulations, we provide in-depth analysis to reveal structural signatures of wild and mutant complexes with their bound agonist and antagonist ligands. We constructed intra-protein network graphs to discriminate the global conformational changes induced by mutations. The simulations also allowed us to elucidate the local side-chain dynamical variations in ligand-bound mutant receptors. The data suggest that the mutation in transmembrane V (V194G) drastically disrupts the organization of ligand binding site and causes disorder in the native helical arrangement. Interestingly, the R237L mutation leads to significant rewiring of side-chain contacts in the intracellular loop 3 (site of mutation) and also affects the distant transmembrane topology. Additionally, these mutations lead to compact ICL3 region compared to the wild type, indicating that the receptor would be inaccessible for G protein coupling. Our findings thus reveal unreported structural determinants of the mutated DRD4 receptor and provide a robust framework for design of effective novel drugs.

Discovery, characterization and biological evaluation of a novel (R)-4,4-difluoropiperidine scaffold as dopamine receptor 4 (D4R) antagonists.

Herein, we report the synthesis and structure-activity relationship of a novel series of (R)-4,4-difluoropiperidine core scaffold as dopamine receptor 4 (D4) antagonists. A series of compounds from this scaffold are highly potent against the D4 receptor and selective against the other dopamine receptors. In addition, we were able to confirm the active isomer as the (R)-enantiomer via an X-ray crystal structure.

Variation at the DRD4 locus is associated with wariness and local site selection in urban black swans.

BACKGROUND: Interactions between wildlife and humans are increasing. Urban animals are often less wary of humans than their non-urban counterparts, which could be explained by habituation, adaptation or local site selection. Under local site selection, individuals that are less tolerant of humans are less likely to settle in urban areas. However, there is little evidence for such temperament-based site selection, and even less is known about its underlying genetic basis. We tested whether site selection in urban and non-urban habitats by black swans (Cygnus atratus) was associated with polymorphisms in two genes linked to fear in animals, the dopamine receptor D4 (DRD4) and serotonin transporter (SERT) genes. RESULTS: Wariness in swans was highly repeatable between disturbance events (repeatability = 0.61) and non-urban swans initiated escape from humans earlier than urban swans. We found no inter-individual variation in the SERT gene, but identified five DRD4 genotypes and an association between DRD4 genotype and wariness. Individuals possessing the most common DRD4 genotype were less wary than individuals possessing rarer genotypes. As predicted by the local site selection hypothesis, genotypes associated with wary behaviour were over three times more frequent at the non-urban site. This resulted in moderate population differentiation at DRD4 (FST = 0.080), despite the sites being separated by only 30 km, a short distance for this highly-mobile species. Low population differentiation at neutrally-selected microsatellite loci and the likely occasional migration of swans between the populations reduces the likelihood of local site adaptations. CONCLUSION: Our results suggest that wariness in swans is partly genetically-determined and that wary swans settle in less-disturbed areas. More generally, our findings suggest that site-specific management strategies may be necessary that consider the temperament of local animals.

Homology modeling, molecular dynamic simulation, and docking based binding site analysis of human dopamine (D4) receptor.

Human dopamine D4 receptor is a GPCR target in the treatment of neurological and psychiatric conditions such as schizophrenia and Parkinson's disease. The X-ray structure of this receptor has not been resolved so far. Therefore, a proper 3D structure of D4 could provide a good tool in order to design novel ligands against this target. In this study, homology modeling studies were performed to obtain a reasonable structure of the receptor using known templates. The obtained model was subjected to molecular dynamic simulation within a DPPC membrane system. Some structural features of the receptor such as a conserved disulfide bridge and ionic lock were considered in the modeling experiments. The resulted trajectories of simulation were clustered based on the root mean square deviation of the backbone. Some known ligands and decoys were accordingly docked into the representative frames of each cluster. The best final model was finally selected based on its ability to discriminate between active ligands and inactive decoys (ROC = 0.839). The presented model of human D4 receptor could be a promising starting point in future studies of drug design for the described target.

Synthesis, pharmacological evaluation and molecular modeling studies of triazole containing dopamine D3 receptor ligands.

A series of 2-methoxyphenyl piperazine analogues containing a triazole ring were synthesized and their in vitro binding affinities at human dopamine D2 and D3 receptors were evaluated. Compounds 5b, 5c, 5d, and 4g, demonstrate high affinity for dopamine D3 receptors and moderate selectivity for the dopamine D3 versus D2 receptor subtypes. To further examine their potential as therapeutic agents, their intrinsic efficacy at both D2 and D3 receptors was determined using a forskolin-dependent adenylyl cyclase inhibition assay. Affinity at dopamine D4 and serotonin 5-HT1A receptors was also determined. In addition, information from previous molecular modeling studies of the binding of a panel of 163 structurally-related benzamide analogues at dopamine D2 and D3 receptors was applied to this series of compounds. The results of the modeling studies were consistent with our previous experimental data. More importantly, the modeling study results explained why the replacement of the amide linkage with the hetero-aromatic ring leads to a reduction in the affinity of these compounds at D3 receptors.

Structure and dynamics of DRD4 bound to an agonist and an antagonist using in silico approaches.

Human dopamine receptor D4 (DRD4), a member of G-protein coupled receptor (GPCR) family, plays a central role in cell signaling and trafficking. Dysfunctional activity of DRD4 can lead to several psychiatric conditions and, therefore, represents target for many neurological disorders. However, lack of atomic structure impairs our understanding of the mechanism regulating its activity. Here, we report the modeled structure of DRD4 alone and in complex with dopamine and spiperone, its natural agonist and antagonist, respectively. To assess the conformational dynamics induced upon ligand binding, all-atom explicit solvent molecular dynamics simulations in membrane environment were performed. Comprehensive analyses of simulations reveal that agonist binding triggers a series of conformational changes in the transmembrane region, including rearrangement of residues, characteristic of transmission and tyrosine toggle molecular switches. Further, the trajectories indicate that a loop region in the intracellular region--ICL3, is significantly dynamic in nature, mainly due to the side-chain movements of conserved proline residues involved in SH3 binding domains. Interestingly, in dopamine-bound receptor simulation, ICL3 represents an open conformation ideal for G protein binding. The structural and dynamical information presented here suggest a mode of activation of DRD4, upon ligand binding. Our study will help in further understanding of receptor activation, as acquiring structural information is crucial for the design of highly selective DRD4 ligands.

Role of dimerization in dopamine D(4) receptor biogenesis.

Dopamine receptors are G protein-coupled receptors critically involved in locomotion, reward, and cognitive processes. Export of dopamine receptors to the plasma membrane is thought to follow the default secretory pathway, whereby proteins travel from the endoplasmatic reticulum (ER), through the Golgi apparatus, to arrive at the cell surface. Several observations indicate that trafficking from the ER to the plasma membrane is tightly regulated, and that correct folding in the ER acts as a bottle neck to the maturation of the dopamine D4 receptors. The dopamine D(4) receptor is an interesting receptor since it has a polymorphic region in its third intracellular loop, resulting in receptor isoforms of varying length and amino acid composition. Correct folding is enhanced by: (1) interaction with specific proteins, such as ER resident chaperones, (2) interaction with pharmacological chaperones, for example, ligands that are membrane permeable and can bind to the receptor in the ER, and (3) receptor dimerization; the assembly of multisubunit proteins into a quaternary structure is started in the ER before cell surface delivery, which helps in correct folding and subsequent expression. These interactions help the process of GPCR folding, but more importantly they ensure that only properly folded proteins proceed from the ER to the trans-Golgi network. In this review we will mainly focus on the role of receptor dimerization in dopamine D(4) receptor maturation.

Dopamine receptor genes and evolutionary differentiation in the domestication of fighting cocks and long-crowing chickens.

The chicken domestication process represents a typical model of artificial selection, and gives significant insight into the general understanding of the influence of artificial selection on recognizable phenotypes. Two Japanese domesticated chicken varieties, the fighting cock (Shamo) and the long-crowing chicken (Naganakidori), have been selectively bred for dramatically different phenotypes. The former has been selected exclusively for aggressiveness and the latter for long crowing with an obedient sitting posture. To understand the particular mechanism behind these genetic changes during domestication, we investigated the degree of genetic differentiation in the aforementioned chickens, focusing on dopamine receptor D2, D3, and D4 genes. We studied other ornamental chickens such as Chabo chickens as a reference for comparison. When genetic differentiation was measured by an index of nucleotide differentiation (NST) newly devised in this study, we found that the NST value of DRD4 for Shamo (0.072) was distinctively larger than those of the other genes among the three populations, suggesting that aggressiveness has been selected for in Shamo by collecting a variety of single nucleotide polymorphisms. In addition, we found that in DRD4 in Naganakidori, there is a deletion variant of one proline at the 24th residue in the repeat of nine prolines of exon 1. We thus conclude that artificial selection has operated on these different kinds of genetic variation in the DRD4 genes of Shamo and Naganakidori so strongly that the two domesticated varieties have differentiated to obtain their present opposite features in a relatively short period of time.

Design, synthesis, lipophilic properties, and binding affinities of potential ligands in positron emission tomography (PET) for visualization of brain dopamine D4 receptors.

We report the synthesis of compounds structurally related to the high-affinity dopamine D4 receptor ligand N-{2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-methoxybenzamide (1e). All compounds were specifically designed as potential PET radioligands for brain D4 receptor visualization, having lipophilicity within a range for brain uptake and weak non-specific binding (0.75100-fold), but its D4 receptor affinity was suboptimal for imaging of brain D4 receptors (Ki =30 nM).