MS Binding Assays for D1 and D5 Dopamine Receptors

MS Binding Assays are a label‐free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but an unlabeled reporter ligand is used instead of a radioligand. The study presented herein describes the development of MS Binding Assays that address D₁ and D₅ dopamine receptors. A highly sensitive, rapid and robust LC–ESI‐MS/MS quantification method for the selective D₁ dopamine receptor antagonist SCH23390 ((5R)‐8‐chloro‐3‐methyl‐5‐phenyl‐1,2,4,5‐tetrahydro‐3‐benzazepin‐7‐ol) was established and validated, using its 8‐bromo analogue SKF83566 as an internal standard. This quantification method proved to be suitable for the characterization of SCH23390 binding to human D₁ and D₅ receptors. Following the concept of MS Binding Assays, saturation experiments for D₁ and D₅ receptors were performed, as well as competition experiments for D₁ receptors. The results obtained are in good agreement with results from radioligand binding assays and therefore indicate that the established MS Binding Assays addressing D₁ and D₅ receptors are well‐suited substitutes for radioligand binding assays, the technique that has so far dominated affinity determinations toward these targets.     

Structure‐Based Virtual Screening for Dopamine D2 Receptor Ligands as Potential Antipsychotics

Structure‐based virtual screening using a D₂ receptor homology model was performed to identify dopamine D₂ receptor ligands as potential antipsychotics. From screening a library of 6.5 million compounds, 21 were selected and were subjected to experimental validation. From these 21 compounds tested, ten D₂ ligands were identified (47.6 % success rate, among them D₂ receptor antagonists, as expected) that have additional affinity for other receptors tested, in particular 5‐HT_2A receptors. The affinity (K_i values) of the compounds ranged from 58 nm to about 24 μm . Similarity and fragment analysis indicated a significant degree of structural novelty among the identified compounds. We found one D₂ receptor antagonist that did not have a protonatable nitrogen atom, which is a key structural element of the classical D₂ pharmacophore model necessary for interaction with the conserved Asp(3.32) residue. This compound exhibited greater than 20‐fold binding selectivity for the D₂ receptor over the D₃ receptor. We provide additional evidence that the amide hydrogen atom of this compound forms a hydrogen bond with Asp(3.32), as determined by tests of its derivatives that cannot maintain this interaction.     

Synthesis, 3D‐QSAR,and Structural Modeling of Benzolactam Derivatives with Binding Affinity for the D2 and D3 Receptors

A series of 37 benzolactam derivatives were synthesized, and their respective affinities for the dopamine D₂ and D₃ receptors evaluated. The relationships between structures and binding affinities were investigated using both ligand‐based (3D‐QSAR) and receptor‐based methods. The results revealed the importance of diverse structural features in explaining the differences in the observed affinities, such as the location of the benzolactam carbonyl oxygen, or the overall length of the compounds. The optimal values for such ligand properties are slightly different for the D₂ and D₃ receptors, even though the binding sites present a very high degree of homology. We explain these differences by the presence of a hydrogen bond network in the D₂ receptor which is absent in the D₃ receptor and limits the dimensions of the binding pocket, causing residues in helix 7 to become less accessible. The implications of these results for the design of more potent and selective benzolactam derivatives are presented and discussed.     

Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

Herein we report the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective agonists of the dopamine‐3 (D₃) receptor. A number of these new compounds bind to the D₃ receptor with sub‐nanomolar affinity and show excellent selectivity (>10 000) for the D₃ receptor over the D₁ and D₂ receptors. For example, compound 23 (N‐(cis‐3‐(2‐(((S)‐2‐amino‐4,5,6,7‐tetrahydrobenzo[d]thiazol‐6‐yl)(propyl)amino)ethyl)‐3‐hydroxycyclobutyl)‐3‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)benzamide) binds to the D₃ receptor with a K_i value of 0.53 nM and shows a selectivity of >20 000 over the D₂ and D₁ receptors in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes. Moreover, in vitro functional assays showed it to be a full agonist for the human D₃ receptor.     

Selective Pharmacophore Models of Dopamine D1 and D2 Full Agonists Based on Extended Pharmacophore Features

This study is focused on the identification of structural features that determine the selectivity of dopamine receptor agonists toward D₁ and D₂ receptors. Selective pharmacophore models were developed for both receptors. The models were built by using projected pharmacophoric features that represent the main agonist interaction sites in the receptor (the Ser residues in TM5 and the Asp in TM3), a directional aromatic feature in the ligand, a feature with large positional tolerance representing the positively charged nitrogen in the ligand, and sets of excluded volumes reflecting the shapes of the receptors. The sets of D₁ and D₂ ligands used for modeling were carefully selected from published sources and consist of structurally diverse, conformationally rigid full agonists as active ligands together with structurally related inactives. The robustness of the models in discriminating actives from inactives was tested against four ensembles of conformations generated by using different established methods and different force fields. The reasons for the selectivity can be attributed to both geometrical differences in the arrangement of the features, e.g., different tilt angels of the π system, as well as shape differences covered by the different sets of excluded volumes. This work provides useful information for the design of new D₁ and D₂ agonists and also for comparative homology modeling of D₁ and D₂ receptors. The approach is general and could therefore be applied to other ligand–protein interactions for which no experimental protein structure is available.     

Mapping the catechol binding site in dopamine D₁ receptors: synthesis and evaluation of two parallel series of bicyclic dopamine analogues

A novel class of isochroman dopamine analogues, originally reported by Abbott Laboratories, have >100‐fold selectivity for D₁‐like over D₂‐like receptors. We synthesized a parallel series of chroman compounds and showed that repositioning the oxygen atom in the heterocyclic ring decreases potency and confers D₂‐like receptor selectivity to these compounds. In silico modeling supports the hypothesis that the altered pharmacology for the chroman series is due to potential intramolecular hydrogen bonding between the oxygen in the chroman ring and the meta‐hydroxy group of the catechol moiety. This interaction realigns the catechol hydroxy groups and disrupts key interactions between these ligands and critical serine residues in TM5 of the D₁‐like receptors. This hypothesis was tested by the synthesis and pharmacological evaluation of a parallel series of carbocyclic compounds. Our results suggest that if the potential for intramolecular hydrogen bonding is removed, D₁‐like receptor potency and selectivity are restored.     

Chiral Aryloxyalkylamines: Selective 5‐HT1B/1D Activation and Analgesic Activity

A series of chiral 2,3‐dichlorophenoxy and 1‐naphthyloxy alkylamines were synthesized, and their binding affinities towards 5‐HT_1D and h5‐HT_1B receptors were evaluated. In the naphthyloxy series, the (R)‐prolinol derivative was the most selective 5‐HT_1D ligand, while (S)‐N‐methyl‐2‐(1‐naphthyloxy)propan‐1‐amine showed the highest selectivity for h5‐HT_1B. Both compounds performed as 5‐HT_1D agonists in the isolated guinea pig assay and showed higher analgesic activity than both sumatriptan and the achiral analogue 20 b in the mouse hot‐plate test. Neither ligand displayed any affinity for nicotinic ACh receptors present in mouse brain membranes, thus indicating that their analgesic activity does not arise through interaction with these receptors.     

Synthesis of Bitopic Ligands as Potent Dopamine D2 Receptor Agonists

In this study, we designed and synthesized twelve bitopic ligands as dopamine D₂ receptor (D₂R) agonists. The forskolin-induced cAMP accumulation assay revealed that all the finial compounds are able to activate D₂R. Furthermore, bitopic ligand N-((trans)-4-(((2,3-dihydro-1H-inden-2-yl)(propyl)amino)methyl)cyclo-hexyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide ( 11 b ) showed 21-fold higher potency than lead compound propyl aminoindane ( 2 ) and 17-fold higher subtype selectivity for D₂R over D₄R, indicating that the optimal length of spacer affects the D₂R functionality. Molecular modeling study exhibited that 11 b formed an electrostatic interaction and two H-bonds with amino acid Asp114, which contributes significantly to the D₂R functional activity. Taken together, we discovered a bitopic ligand 11 b as potent D₂R agonist, which may be used as a tool compound for further study.     

N‐(Anilinoethyl)amides: Design and Synthesis of Metabolically Stable,Selective Melatonin Receptor Ligands

The class of N‐(anilinoethyl)amides includes melatonin receptor ligands with varied subtype selectivity and intrinsic activity. One of these ligands, the MT₂‐selective partial agonist UCM765 (N‐{2‐[(3‐methoxyphenyl)phenylamino]ethyl}acetamide), had evidenced hypnotic effects in rodents at doses ≥40 mg kg^?1 (s.c.), in spite of its sub‐nanomolar affinity for human melatonin receptors. Supposing that its low in vivo potency could be due, at least in part, to metabolic liability in rat liver, UCM765 was incubated with rat liver S9 fraction and rat, mouse, or human microsomes, and the major metabolites were identified by LC–MS, synthesized, and in vitro tested for their affinity toward MT₁ and MT₂ receptors. The obtained information was exploited to design novel analogues of UCM765 that are more resistant to in vitro oxidative degradation, while maintaining a similar binding profile. The analogue UCM924 (N‐{2‐[(3‐bromophenyl)‐(4‐fluorophenyl)amino]ethyl}acetamide) displayed a binding profile similar to that of UCM765 on cloned human receptors (MT₂‐selective partial agonist) and a significantly longer half‐life in the presence of rat liver S9 fraction.     

Switchable Stereoselectivity: The Effects of Substituents on the D2‐Symmetric Biphenyl Backbone of Phosphoramidites in Copper‐Catalyzed Asymmetric Conjugate Addition Reactions with Triethylaluminium

A highly enantioselective copper‐catalyzed conjugate addition with triethylaluminium was developed using phosphoramidite ligands bearing a D₂‐symmetric biphenyl backbone. For these ligands we demonstrated that the 3,3′,5,5′‐substituents on the biphenyl backbone can completely reverse the absolute configuration of the products.     

Carbohydrate–PNA and Aptamer–PNA Conjugates for the Spatial Screening of Lectins and Lectin Assemblies

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA‐programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well‐defined Ricinus communis agglutinin (RCA₁₂₀), and 2) rather ill‐defined assemblies of L‐selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N‐acetyllactosamine (LacNAc), sialyl‐Lewis‐X (sLe^X), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA₁₂₀ was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA₁₂₀ affinity (K_D=4 μM ). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L‐selectin assemblies were probed. This study involved the bivalent display both of the weak‐affinity sLe^X ligand and of a high‐affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self‐assemblies were targeted against L‐selectin on gold nanoparticles. Spatial screening of L‐selectin on the surfaces of leukocytes showed higher affinity enhancements (25‐fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L‐selectin.     

A Single Mutation at the Ferredoxin Binding Site of P450 Vdh Enables Efficient Biocatalytic Production of 25‐Hydroxyvitamin D3

Vitamin D₃ hydroxylase (Vdh) from Pseudonocardia autotrophica is a cytochrome P450 monooxygenase that catalyzes the two‐step hydroxylation of vitamin D₃ (VD₃) to produce 25‐hydroxyvitamin D₃ (25(OH)VD₃) and 1α,25‐dihydroxyvitamin D₃ (1α,25(OH)₂VD₃). These hydroxylated forms of VD₃ are useful as pharmaceuticals for the treatment of conditions associated with VD₃ deficiency and VD₃ metabolic disorder. Herein, we describe the creation of a highly active T107A mutant of Vdh by engineering the putative ferredoxin‐binding site. Crystallographic and kinetic analyses indicate that the T107A mutation results in conformational change from an open to a closed state, thereby increasing the binding affinity with ferredoxin. We also report the efficient biocatalytic synthesis of 25(OH)VD₃, a promising intermediate for the synthesis of various hydroxylated VD₃ derivatives, by using nisin‐treated Rhodococcus erythropolis cells containing Vdh_T107A. The gene‐expression cassette encoding Bacillus megaterium glucose dehydrogenase‐IV was inserted into the R. erythropolis chromosome and expressed to avoid exhaustion of NADH in a cytoplasm during bioconversion. As a result, approximately 573 μg mL^?1 25(OH)VD₃ was successfully produced by a 2 h bioconversion.     

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

The human dopamine receptors D_2S and D₃ belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D_2S and D₃ receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate K_D-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.     

Synthesis,Pharmacological Evaluation,and Docking Studies of Novel Pyridazinone‐Based Cannabinoid Receptor Type 2 Ligands

In recent years, cannabinoid type 2 receptors (CB₂R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB₂R are devoid of the psychoactive effects typically observed for CB₁R ligands. Based on our recent studies on a class of pyridazinone 4‐carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure–activity relationships for this promising scaffold with the aim to develop potent CB₂R ligands. In binding assays, two of the new synthesized compounds [6‐(3,4‐dichlorophenyl)‐2‐(4‐fluorobenzyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2,3‐dihydropyridazine‐4‐carboxamide ( 2 ) and 6‐(4‐chloro‐3‐methylphenyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2‐pentyl‐2,3‐dihydropyridazine‐4‐carboxamide ( 22 )] showed high CB₂R affinity, with K_i values of 2.1 and 1.6 nm , respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB₂R inverse agonists. Compound 22 displayed the highest CB₂R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle‐switch residue, W6.48.     

Bivalent Argininamide‐Type Neuropeptide Y Y1 Antagonists Do Not Support the Hypothesis of Receptor Dimerisation

Bivalent ligands are potential tools to investigate the dimerisation of G‐protein‐coupled receptors. Based on the (R)‐argininamide BIBP 3226, a potent and selective neuropeptide Y Y₁ receptor (Y₁R) antagonist, we prepared a series of bivalent Y₁R ligands with a wide range of linker lengths (8–36 atoms). Exploiting the high eudismic ratio (>1000) of the parent compound, we synthesised sets of R,R‐, R,S‐ and S,S‐configured bivalent ligands to gain insight into the “bridging” of two Y₁Rs by simultaneous interaction with both binding sites of a putative receptor dimer. Except for the S,S isomers, the bivalent ligands are high‐affinity Y₁R antagonists, as determined by Ca²⁺ assays on HEL cells and radioligand competition assays on human Y₁R‐expressing SK‐N‐MC and MCF‐7 cells. Whereas the R,R enantiomers are most potent, no marked differences were observed relative to the corresponding meso forms. The difference between R,R and R,S diastereomers was most pronounced (about sixfold) in the case of the Y₁R antagonist containing a spacer of 20 atoms in length. Among the R,R enantiomers, linker length and structural diversity had little effect on Y₁R affinity. Although the bivalent ligands preferentially bind to the Y₁R, the selectivity toward human Y₂, Y₄, and Y₅ receptors was markedly lower than that of the monovalent argininamides. The results of this study neither support the presence of Y₁R dimers nor the simultaneous occupation of both binding pockets by the twin compounds. However, as the interaction with Y₁R dimers cannot be unequivocally ruled out, the preparation of a bivalent radioligand is suggested to determine the ligand–receptor stoichiometry. Aiming at such radiolabelled pharmacological tools, prototype twin compounds were synthesised, containing an N‐propionylated amino‐functionalised branched linker (K_i≥18 nM ), a tritiated form of which can be easily prepared.     

Structure–Activity Relationships of Quinoxaline‐Based 5‐HT3A and 5‐HT3AB Receptor‐Selective Ligands

Until recently, discriminating between homomeric 5‐HT₃A and heteromeric 5‐HT₃AB receptors was only possible with ligands that bind in the receptor pore. This study describes the first series of ligands that can discriminate between these receptor types at the level of the orthosteric binding site. During a recent fragment screen, 2‐chloro‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline (VUF10166) was identified as a ligand that displays an 83‐fold difference in [³H]granisetron binding affinity between 5‐HT₃A and 5‐HT₃AB receptors. Fragment hit exploration, initiated from VUF10166 and 3‐(4‐methylpiperazin‐1‐yl)quinoxalin‐2‐ol, resulted in a series of compounds with higher affinity at either 5‐HT₃A or 5‐HT₃AB receptors. These ligands reveal that a single atom is sufficient to change the selectivity profile of a compound. At the extremes of the new compounds were 2‐amino‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed 11‐fold selectivity for the 5‐HT₃A receptor, and 2‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed an 8.3‐fold selectivity for the 5‐HT₃AB receptor. These compounds represent novel molecular tools for studying 5‐HT₃ receptor subtypes and could help elucidate their physiological roles.     

Lipocalin‐Type Prostaglandin D Synthase Is a Novel Phytocannabinoid‐Binding Protein

Lipocalin‐type prostaglandin D synthase (L‐PGDS; EC:5.3.99.2) is an enzyme with dual functional roles as a prostaglandin D₂‐synthesizing enzyme and as an extracellular transporter for diverse lipophilic compounds in the cerebrospinal fluid (CSF). Transport of hydrophobic endocannabinoids is mediated by serum albumin in the blood and intracellularly by the fatty acid binding proteins, but no analogous transport mechanism has yet been described in CSF. L‐PGDS has been reported to promiscuously bind a wide variety of lipophilic ligands and is among the most abundant proteins found in the CSF. Here, we examine the binding of several classes of endogenous and synthetic ligands to L‐PGDS. Endocannabinoids exhibited low affinity toward L‐PGDS, while cannabinoid metabolites and synthetic cannabinoids displayed higher affinities for L‐PGDS. These results indicate that L‐PGDS is unlikely to function as a carrier for endocannabinoids in the CSF, but it may bind and transport a subset of cannabinoids.     

Insight into the Interactions between Novel Coumarin Derivatives and Human A3 Adenosine Receptors

A study focused on the discovery of new chemical entities based on the 3‐arylcoumarin scaffold was performed with the aim of finding new adenosine receptor (AR) ligands. Thirteen synthesized compounds were evaluated by radioligand binding (A₁, A_2A, and A₃) and adenylyl cyclase activity (A_2B) assays in order to study their affinity for the four human AR (hAR) subtypes. Seven of the studied compounds proved to be selective A₃AR ligands, with 3‐(4′‐methylphenyl)‐8‐(2‐oxopropoxy)coumarin ( 12 ) being the most potent (K_i=634 nM ). None of the compounds showed affinity for the A_2B receptor, while four compounds were found to be nonselective AR ligands for the other three subtypes. Docking simulations were carried out to identify the hypothetical binding mode and to rationalize the interaction of these types of coumarin derivatives with the binding site of the three ARs to which binding was observed. The results allowed us to conclude that the 3‐arylcoumarin scaffold composes a novel and promising class of A₃AR ligands. ADME properties were also calculated, with the results suggesting that these compounds are promising leads for the identification of new drug candidates.     

GluN2B‐Selective N‐Methyl‐d‐aspartate (NMDA) Receptor Antagonists Derived from 3‐Benzazepines: Synthesis and Pharmacological Evaluation of Benzo[7]annulen‐7‐amines

Given their high neuroprotective potential, ligands that block GluN2B‐containing N‐methyl‐D ‐aspartate (NMDA) receptors by interacting with the ifenprodil binding site located on the GluN2B subunit are of great interest for the treatment of various neuronal disorders. In this study, a novel class of GluN2B‐selective NMDA receptor antagonists with the benzo[7]annulene scaffold was prepared and pharmacologically evaluated. The key intermediate, N‐(2‐methoxy‐5‐oxo‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐yl)acetamide ( 11 ), was obtained by cyclization of 3‐acetamido‐5‐(3‐methoxyphenyl)pentanoic acid ( 10 b ). The final reaction steps comprise hydrolysis of the amide, reduction of the ketone, and reductive alkylation, leading to cis‐ and trans‐configured 7‐(ω‐phenylalkylamino)benzo[7]annulen‐5‐ols. High GluN2B affinity was observed with cis‐configured γ‐amino alcohols substituted with a 3‐phenylpropyl moiety at the amino group. Removal of the benzylic hydroxy moiety led to the most potent GluN2B antagonists of this series: 2‐methoxy‐N‐(3‐phenylpropyl)‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐amine ( 20 a , K_i=10 nM ) and 2‐methoxy‐N‐methyl‐N‐(3‐phenylpropyl)‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐amine ( 23 a , K_i=7.9 nM ). The selectivity over related receptors (phencyclidine binding site of the NMDA receptor, σ₁ and σ₂ receptors) was recorded. In a functional assay measuring the cytoprotective activity of the benzo[7]annulenamines, all tested compounds showed potent NMDA receptor antagonistic activity. Cytotoxicity induced via GluN2A subunit‐containing NMDA receptors was not inhibited by the new ligands.