Structure–Activity Relationship and Evaluation of Phenethylamine and Tryptamine Derivatives for Affinity towards 5-Hydroxytryptamine Type 2A Receptor

Among 14 subtypes of serotonin receptors (5-HTRs), 5-HT 2AR plays important roles in drug addiction and various psychiatric disorders. Agonists for 5-HT 2AR have been classified into three structural groups: phenethylamines, tryptamines, and ergolines. In this study, the structure-activity relationship (SAR) of phenethylamine and tryptamine derivatives for binding 5-HT 2AR was determined. In addition, functional and regulatory evaluation of selected compounds was conducted for extracellular signal-regulated kinases (ERKs) and receptor endocytosis. SAR studies showed that phenethylamines possessed higher affinity to 5-HT 2AR than tryptamines. In phenethylamines, two phenyl groups were attached to the carbon and nitrogen (R 3 ) atoms of ethylamine, the backbone of phenethylamines. Alkyl or halogen groups on the phenyl ring attached to the β carbon exerted positive effects on the binding affinity when they were at para positions. Oxygen-containing groups attached to R 3 exerted mixed influences depending on the position of their attachment. In tryptamine derivatives, tryptamine group was attached to the β carbon of ethylamine, and ally groups were attached to the nitrogen atom. Oxygen-containing substituents on large ring and alkyl substituents on the small ring of tryptamine groups exerted positive and negative influence on the affinity for 5-HT 2AR, respectively. Ally groups attached to the nitrogen atom of ethylamine exerted negative influences. Functional and regulatory activities of the tested compounds correlated with their affinity for 5-HT 2AR, suggesting their agonistic nature. In conclusion, this study provides information for designing novel ligands for 5-HT 2AR, which can be used to control psychiatric disorders and drug abuse.

Potential Functional Role of Phenethylamine Derivatives in Inhibiting Dopamine Reuptake: Structure–Activity Relationship

Numerous psychotropic and addictive substances possess structural features similar to those of β-phenethylamine (β-PEA). In this study, we selected 29 β-PEA derivatives and determined their structure–activity relationship (SAR) to their ability to inhibit dopamine (DA) reuptake; conducted docking simulation for two selected compounds; and identified their potential functionals. The compounds were subdivided into arylethylamines, 2-(alkyl amino)-1-arylalkan-1-one derivatives and alkyl 2-phenyl-2-(piperidin-2-yl)acetate derivatives. An aromatic group, alkyl group, and alkylamine derivative were attached to the arylethylamine and 2-(alkyl amino)-1-arylalkan-1-one derivatives. The inhibitory effect of the compounds on dopamine reuptake increased in the order of the compounds substituted with phenyl, thiophenyl, and substituted phenyl groups in the aromatic position; compounds with longer alkyl groups and smaller ring-sized compounds at the alkylamine position showed stronger inhibitory activities. Docking simulation conducted for two compounds, 9 and 28, showed that the (S)-form of compound 9 was more stable than the (R)-form, with a good fit into the binding site covered by helices 1, 3, and 6 of human dopamine transporter (hDAT). In contrast, the (R, S)-configuration of compound 28 was more stable than that of other isomers and was firmly placed in the binding pocket of DAT bound to DA. DAinduced endocytosis of dopamine D2 receptors was inhibited when they were co-expressed with DAT, which lowered extracellular DA levels, and uninhibited when they were pretreated with compound 9 or 28. In summary, this study revealed critical structural features responsible for the inhibition of DA reuptake and the functional role of DA reuptake inhibitors in regulating D2 receptor function.

Design, Synthesis, and Functional Evaluation of 1, 5-Disubstituted Tetrazoles as Monoamine Neurotransmitter Reuptake Inhibitors

Tetrazoles were designed and synthesized as potential inhibitors of triple monoamine neurotransmitters (dopamine, norepinephrine, serotonin) reuptake based on the functional and docking simulation of compound 6 which were performed in a previous study. The compound structure consisted of a tetrazole-linker (n)-piperidine/piperazine-spacer (m)-phenyl ring, with tetrazole attached to two phenyl rings (R1 and R2). Altering the carbon number in the linker (n) from 3 to 4 and in the spacer (m) from 0 to 1 increased the potency of serotonin reuptake inhibition. Depending on the nature of piperidine/piperazine, the substituents at R1 and R2 exerted various effects in determining their inhibitory effects on monoamine reuptake. Docking study showed that the selectivity of tetrazole for different transporters was determined based on multiple interactions with various residues on transporters, including hydrophobic residues on transmembrane domains 1, 3, 6, and 8. Co-expression of dopamine transporter, which lowers dopamine concentration in the biophase by uptaking dopamine into the cells, inhibited the dopamine-induced endoctytosis of dopamine D2 receptor. When tested for compound 40 and 56, compound 40 which has more potent inhibitory activity on dopamine reuptake more strongly disinhibited the inhibitory activity of dopamine transporter on the endocytosis of dopamine D2 receptor. Overall, we identified candidate inhibitors of triple monoamine neurotransmitter reuptake and provided a theoretical background for identifying such neurotransmitter modifiers for developing novel therapeutic agents of various neuropsychiatric disorders.

Structural Requirements for Modulating 4-Benzylpiperidine Carboxamides from Serotonin/Norepinephrine Reuptake Inhibitors to Triple Reuptake Inhibitors

In this study, we determined the effect of 24 different synthetic 4-benzylpiperidine carboxamides on the reuptake of serotonin, norepinephrine, and dopamine (DA), and characterized their structure–activity relationship. The compounds with a two-carbon linker inhibited DA reuptake with much higher potency than those with a three-carbon linker. Among the aromatic ring substituents, biphenyl and diphenyl groups played a critical role in determining the selectivity of the 4-benzylpiperidine carboxamides toward the serotonin transporter (SERT) and dopamine transporter (DAT), respectively. Compounds with a 2-naphthyl ring were found to exhibit a higher degree of inhibition on the norepinephrine transporter (NET) and SERT than those with a 1-naphthyl ring. A docking simulation using a triple reuptake inhibitor 8k and a serotoninorepinephrine reuptake inhibitor 7j showed that the regions spanning transmembrane domain (TM)1, TM3, and TM6 form the ligand binding pocket. The compound 8k bound tightly to the binding pocket of all three monoamine reuptake transporters; however, 7j showed poor docking with DAT. Co-expression of DAT with the dopamine D2 receptor (D2R) significantly inhibited DA-induced endocytosis of D2R probably by reuptaking DA into the cells. Pretreatment of the cells with 8f, which is one of the compounds with good inhibitory activity on DAT, blocked DAT-induced inhibition of D2R endocytosis. In summary, this study identified critical structural features contributing to the selectivity of a molecule for each of the monoamine transporters, critical residues on the compounds that bound to the transporters, and the functional role of a DA reuptake inhibitor in regulating D2R function.

Structural Requirements for Modulating 4-Benzylpiperidine Carboxamides from Serotonin/Norepinephrine Reuptake Inhibitors to Triple Reuptake Inhibitors

In this study, we determined the effect of 24 different synthetic 4-benzylpiperidine carboxamides on the reuptake of serotonin, norepinephrine, and dopamine (DA), and characterized their structure–activity relationship. The compounds with a two-carbon linker inhibited DA reuptake with much higher potency than those with a three-carbon linker. Among the aromatic ring substituents, biphenyl and diphenyl groups played a critical role in determining the selectivity of the 4-benzylpiperidine carboxamides toward the serotonin transporter (SERT) and dopamine transporter (DAT), respectively. Compounds with a 2-naphthyl ring were found to exhibit a higher degree of inhibition on the norepinephrine transporter (NET) and SERT than those with a 1-naphthyl ring. A docking simulation using a triple reuptake inhibitor 8k and a serotoninorepinephrine reuptake inhibitor 7j showed that the regions spanning transmembrane domain (TM)1, TM3, and TM6 form the ligand binding pocket. The compound 8k bound tightly to the binding pocket of all three monoamine reuptake transporters; however, 7j showed poor docking with DAT. Co-expression of DAT with the dopamine D2 receptor (D2R) significantly inhibited DA-induced endocytosis of D2R probably by reuptaking DA into the cells. Pretreatment of the cells with 8f, which is one of the compounds with good inhibitory activity on DAT, blocked DAT-induced inhibition of D2R endocytosis. In summary, this study identified critical structural features contributing to the selectivity of a molecule for each of the monoamine transporters, critical residues on the compounds that bound to the transporters, and the functional role of a DA reuptake inhibitor in regulating D2R function.

Catalpol and Mannitol, Two Components of Rehmannia glutinosa, Exhibit Anticonvulsant Effects Probably via GABA A Receptor Regulation

Epilepsy is a brain disorder that affects millions of people worldwide and is usually managed using currently available antiepileptic drugs, which result in adverse effects and are ineffective in approximately 20–25% of patients. Thus, there is growing interest in the development of new antiepileptic drugs with fewer side effects. In a previous study, we showed that a Rehmannia glutinosa (RG) water extract has protective effects against electroshock- and pentylenetetrazol (PTZ)-induced seizures, with fewer side effects. In this study, the objective was to identify the RG components that are responsible for its anticonvulsant effects. Initially, a number of RG components (aucubin, acteoside, catalpol, and mannitol) were screened, and the anticonvulsant effects of different doses of catalpol, mannitol, and their combination on electroshock- and chemically (PTZ or strychnine)-induced seizures in mice, were further assessed. Gamma-aminobutyric acid (GABA) receptor binding assay and electroencephalography (EEG) analysis were conducted to identify the potential underlying drug mechanism. Additionally, treated mice were tested using open-field and rotarod tests. Catalpol, mannitol, and their combination increased threshold against electroshock-induced seizures, and decreased the percentage of seizure responses induced by PTZ, a GABA antagonist. GABA receptor binding assay results revealed that catalpol and mannitol are associated with GABA receptor activity, and EEG analysis provided evidence that catalpol and mannitol have anticonvulsant effects against PTZ-induced seizures. In summary, our results indicate that catalpol and mannitol have anticonvulsant properties, and may mediate the protective effects of RG against seizures.

Role of Helix 8 in Dopamine Receptor Signaling

G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished β-arrestin-mediated desensitization, resulting in increased Gs signaling.

Conceptual Progress for the Improvements in the Selectivity and Efficacy of G Protein-Coupled Receptor Therapeutics: An Overview

No abstract available.

Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis.

Molecular Signature That Determines the Acute Tolerance of G Protein-Coupled Receptors

Desensitization and acute tolerance are terms used to describe the attenuation of receptor responsiveness by prolonged or intermittent exposure to an agonist. Unlike desensitization of G protein-coupled receptors (GPCRs), which is commonly explained by steric hindrance caused by the β-arrestins that are translocated to the activated receptors, molecular mechanisms involved in the acute tolerance of GPCRs remain unclear. Our studies with several GPCRs and related mutants showed that the acute tolerance of GPCRs could occur independently of agonist-induced β-arrestin translocation. A series of co-immunoprecipitation experiments revealed a correlation between receptor tolerance and interactions among receptors, β-arrestin2, and Gβγ. Gβγ displayed a stable interaction with receptors and β-arrestin2 in cells expressing GPCRs that were prone to undergo tolerance compared to the GPCRs that were resistant to acute tolerance. Strengthening the interaction between Gβγ and β-arrestin rendered the GPCRs to acquire the tendency of acute tolerance. Overall, stable interaction between the receptor and Gβγ complex is required for the formation of a complex with β-arrestin, and determines the potential of a particular GPCR to undergo acute tolerance. Rather than turning off the signal, β-arrestins seem to contribute on continuous signaling when they are in the context of complex with receptor and Gβγ.

Functional Regulation of Dopamine D₃ Receptor through Interaction with PICK1

PICK1, a PDZ domain-containing protein, is known to increase the reuptake activities of dopamine transporters by increasing their expressions on the cell surface. Here, we report a direct and functional interaction between PICK1 and dopamine D₃ receptors (D₃R), which act as autoreceptors to negatively regulate dopaminergic neurons. PICK1 colocalized with both dopamine D₂ receptor (D₂R) and D₃R in clusters but exerted different functional influences on them. The cell surface expression, agonist affinity, endocytosis, and signaling of D₂R were unaffected by the coexpression of PICK1. On the other hand, the surface expression and tolerance of D₃R were inhibited by the coexpression of PICK1. These findings show that PICK1 exerts multiple effects on D₃R functions.

Roles of Dopamine D₂ Receptor Subregions in Interactions with β-Arrestin2

β-Arrestins are one of the protein families that interact with G protein-coupled receptors (GPCRs). The roles of β-arrestins are multifaceted, as they mediate different processes including receptor desensitization, endocytosis, and G protein-independent signaling. Thus, determining the GPCR regions involved in the interactions with β-arrestins would be a preliminary step in understanding the molecular mechanisms involved in the selective direction of each function. In the current study, we determined the roles of the N-terminus, intracellular loops, and C-terminal tail of a representative GPCR in the interaction with β-arrestin2. For this, we employed dopamine D₂ and D₃ receptors (D₂R and D₃R, respectively), since they display distinct agonist-induced interactions with β-arrestins. Our results showed that the second and third intracellular loops of D₂R are involved in the agonist-induced translocation of β-arrestins toward plasma membranes. In contrast, the N- and C-termini of D₂R exerted negative effects on the basal interaction with β-arrestins.