Selectivity and activation of dopamine D3R from molecular dynamics

D₃ receptor, a member of dopamine (DA) D₂-like receptor family, which belongs to class A of G-protein coupled receptors (GPCRs), has been reported to play a critical role in neuropsychiatric disorders. Recently, the crystal structure of human dopamine D3 receptor was reported, which facilitates structure-based drug discovery of D3R significantly. We dock D3R-selective compounds into the crystal structure of D3R and homology structure of D2R. Then we perform 20?ns molecular dynamics (MD) of the receptor with selective compounds bound in explicit lipid and water. Our docking and MD results indicate the important residues related to the selectivity of D3R. Specifically, residue Thr^7.39 in D3R may contribute to the high selectivity of R-22 with D3R. Meanwhile, the 4-carbon linker and phenylpiperazine of R-22 improve the binding affinity and the selectivity with D3R. We also dock the agonists, including dopamine, into D3R and perform MD. Our molecular dynamics results of D3R with agonist bound show strong conformational changes from TM5, TM6, and TM7, outward movement of intracellular part of TM6, fluctuation of “ionic lock” motif and conformational change of Tyr^7.53, which is consistent with recent crystal structures of active GPCRs and illustrates the dynamical process during activation. Our results reveal the mechanism of selectivity and activation for D3R, which is important for developing high selective antagonists and agonists for D3R.     

Synthesis and SAR of aminothiazole fused benzazepines as selective dopamine D2 partial agonists

Dopamine (D₂) partial agonists (D2PAs) have been regarded as a potential treatment for schizophrenia patients with expected better side effect profiles than currently marketed antipsychotics. Herein we report the synthesis and SAR of a series of aminothiazole fused benzazepines as selective D₂ partial agonists. These compounds have good selectivity, CNS drug-like properties and tunable D₂ partial agonism. One of the key compounds, 8h, has good in vitro/in vivo ADME characteristics, and is active in a rat amphetamine-induced locomotor activity model.     

3,4-Dihydroxy- and 3,4-methylenedioxy- phenanthrene-type alkaloids with high selectivity for D2 dopamine receptor

Dopamine-mediated neurotransmission plays an important role in relevant psychiatric and neurological disorders. Nowadays, there is an enormous interest in the development of new drugs acting at the dopamine receptors (DR) as potential new targets for the treatment of schizophrenia or Parkinson’s disease. Previous studies have revealed that isoquinoline compounds such as tetrahydroisoquinolines (THIQs) can behave as selective D₂ dopaminergic alkaloids. In the present study we have synthesized five aporphine compounds and five phenanthrene alkaloids and evaluated their potential dopaminergic activity. Binding studies on rat striatal membranes were used to evaluate their affinity and selectivity towards D₁ and D₂ DR. Phenanthrene type alkaloids, in particular the 3,4-dihydroxy- and 3,4-methylenedioxy derivatives, displayed high selectivity towards D₂ DR. Therefore, they are potential candidates to be used in the treatment of schizophrenia (antagonists) or Parkinson’s disease (agonists) due to their scarce D₁ DR-associated side effects.     

Discovery of new SCH 39166 analogs as potent and selective dopamine D1 receptor antagonists

A series of novel dopamine D₁ antagonists derived from functionalization of the D-ring of SCH 39166 were prepared. A number of these compounds displayed subnanomolar D₁ activity and more than 1000-fold selectivity over D₂. We found C-3 derivatization afforded compounds with superior overall profile in comparison to the C-2 and C-4 derivatization. A number of highly potent D₁ antagonists were discovered which have excellent selectivity over other dopamine receptors and improved PK profile compared to SCH 39166.     

Remote functionalization of SCH 39166: Discovery of potent and selective benzazepine dopamine D1 receptor antagonists

A series of novel benzazepine derived dopamine D₁ antagonists have been discovered. These compounds are highly potent at D₁ and showed excellent selectivity over D₂ and D₄ receptors. SAR studies revealed that a variety of functional groups are tolerated on the D-ring of known tetracyclic benzazepine analog 2, SCH 39166, leading to compounds with nanomolar potency at D₁ and good selectivity over D₂-like receptors.     

Design and synthesis of novel N-sulfonyl-2-indoles that behave as 5-HT6 receptor ligands with significant selectivity for D3 over D2 receptors

All clinically-used antipsychotics display similar affinity for both D₂ (D2R) and D₃ (D3R) receptors, and they likewise act as 5-HT_2A receptor antagonists. They provide therapeutic benefit for positive symptoms, but no marked or consistent improvement in neurocognitive, social cognitive or negative symptoms. Since blockade of D₃ and 5-HT₆ (5-HT6R) receptors enhances neurocognition and social cognition, and potentially improves negative symptoms, a promising approach for improved treatment for schizophrenia would be to develop drugs that preferentially act at D3R versus D2R and likewise recognize 5-HT6R. Starting from the high affinity 5-HT6R ligands I and II, we identified compounds 11a and 14b that behave as 5-HT6R ligands with significant selectivity for D3R over D2R.     

1-substituted apomorphines as potent dopamine agonists

A novel set of 1-substituted apomorphines as dopaminergic agonists were synthesized according to our new strategy employing the acid-catalyzed rearrangement of diversely functionalized 5β-substituted-6-demethoxythebaines. The activities of new compounds for dopamine receptors subtypes were evaluated using HEK293 based stable cell lines expressing D₁, D_2L or D₃ receptor subtypes. All studied compounds had affinities in nanomolar range for D_2L and D₃ receptors and the change of the nature of substituent in position 1 had only moderate effect. D₁ receptors were sensitive to the introduction of the 4-OH-benzyl function resulting in an increased affinity. The small hydrophilic group (hydroxymethyl) highly reduced the agonist affinity and potency thereby increasing subtype selectivity. This strategy for selective modulation of affinities and potencies of 1-substituted apomorphines gives essential hints for future design of subtype selective dopaminergic ligands.     

Receptor,Ligand and Transducer Contributions to Dopamine D2 Receptor Functional Selectivity

Functional selectivity (or biased agonism) is a property exhibited by some G protein-coupled receptor (GPCR) ligands, which results in the modulation of a subset of a receptor’s signaling capabilities and more precise control over complex biological processes. The dopamine D2 receptor (D₂R) exhibits pleiotropic responses to the biogenic amine dopamine (DA) to mediate complex central nervous system functions through activation of G proteins and β-arrestins. D₂R is a prominent therapeutic target for psychological and neurological disorders in which DA biology is dysregulated and targeting D₂R with functionally selective drugs could provide a means by which pharmacotherapies could be developed. However, factors that determine GPCR functional selectivity in vivo may be multiple with receptors, ligands and transducers contributing to the process. We have recently described a mutagenesis approach to engineer biased D₂R mutants in which G protein-dependent (^[Gprot]D₂R) and β-arrestin-dependent signaling (^[βarr]D₂R) were successfully separated (Peterson, et al. PNAS, 2015). Here, permutations of these mutants were used to identify critical determinants of the D₂R signaling complex that impart signaling bias in response to the natural or synthetic ligands. Critical residues identified in generating ^[Gprot]D₂R and ^[βarr]D₂R conferred control of partial agonism at G protein and/or β-arrestin activity. Another set of mutations that result in G protein bias was identified that demonstrated that full agonists can impart unique activation patterns, and provided further credence to the concept of ligand texture. Finally, the contributions and interplay between different transducers indicated that G proteins are not aberrantly activated, and that receptor kinase and β-arrestin activities are inextricably linked. These data provide a thorough elucidation of the feasibility and malleability of D₂R functional selectivity and point to means by which novel in vivo therapies could be modeled.     

Novel N-biphenyl-2-ylmethyl 2-methoxyphenylpiperazinylalkanamides as 5-HT7R antagonists for the treatment of depression

5-HT₇ receptor (5-HT₇R) is a promising target for the treatment of depression and neuropathic pain. 5-HT₇R antagonists exhibited antidepressant effects, while the agonists produced strong anti-hyperalgesic effects. In our efforts to discover selective 5-HT₇R antagonists or agonists, N-biphenylylmethyl 2-methoxyphenylpiperazinylalkanamides 1 were designed, synthesized, and biologically evaluated against 5-HT₇R. Among the synthesized compounds, N-2′-chlorobiphenylylmethyl 2-methoxyphenylpiperazinylpentanamide 1–8 showed the best binding affinity with a K_i value of 8.69 nM and it was verified as a novel antagonist according to functional assays. The compound 1–8 was very selective over 5-HT_1DR, 5-HT_2AR, 5-HT₃R, 5-HT_5AR and 5-HT₆R and moderately selective over 5-HT_1AR, 5-HT_1BR and 5-HT_2CR. The novel 5-HT₇R antagonist 1–8 exhibited an antidepressant effect at a dose of 25 mg/kg in the forced swimming test in mice and showed a U-shaped dose–response curve which typically appears in 5-HT₇R antagonists such as SB-269970 and lurasidone.     

Dopamine receptors in bovine retina : characterization of the 3H-spiroperidol binding and its use for screening dopamine receptor affinity of drugs.

³H-spiroperidol bound in a saturable, stereospecifically displaceable manner to homogenates of bovine retina. Scatchard analysis of saturation experiments showed a K_D of 1.35 nM and a density of binding sites of 107 fmoles · mg protein^?1. Stereospecifically displaceable binding was pH and temperature dependent and linear with tissue concentration. Spiroperidol, pimozide, haloperidol and d-butaclamol were the most potent compounds in drug displacement curves (8.74 > pIC₅₀ > 7.61 M). Other neuroleptics such as cisflupenthixol, fluphenazine, clozapine, chlorpromazine and pipamperone, were one order of magnitude less potent. Among dopamine agonists, apomorphine (pIC₅₀ = 7.08 ± 0.19 M) was about 50 times more potent than dopamine itself, epinine and ADTN. Serotonin, α- and ß-adrenergic receptors agonists and antagonists were inactive. These results indicate that the sites labelled by ³H-spiroperidol in retina are dopaminergic; moreover the rank order of various antagonists and agonists observed in displacement curves suggests that this preparation could also provide a useful tool to reveal the selective affinity of drugs for the CNS dopamine receptor linked to the enzyme adenylylcyclase (D₁-receptors).     

S-(+)-aporphines are not selective for human D3 dopamine receptors

Summary 1. Our aim was to test the hypothesis that selectivity for D₃ dopamine (DA) receptors may contribute to limbic anti-DA selectivity ofS-(+)-aporphine DA partial agonists.2. Affinity was tested with³H-emonapride, using human D₃ receptors in mouse fibroblasts and D₂ receptors in rat striatal tissue.3. D₃ receptors showed a picomolar affinity for³H-emonapride, Na⁺ dependence, and reversible saturability, as well as stereoselectivity. Confirmatory or novel D₃/D₂ pharmacologic selectivity was found with several benzamides, thioxanthenes, buspirone, GBR-12909, and DA agonists including hydroxyaminotetralins [ADTN, (+)-7-OH-DPAT, (–)-PPHT and its fluorescein derivative], (–)-N-propylnorapomorphine, (–)-3-PPP, (–)-quinpirole, and SDZ-205-502, but neither aminoergoline nor (+)-aporphine partial agonists.4. The results extend pharmacologic characterization of D₃-transfected cell membranes but fail to account for the high limbic anti-DA selectivity ofS-(+)-aporphines.     

Pre- and Postnatal Ontogeny and Characterization of Dopaminergic D2, Serotonergic S2, and Spirodecanone Binding Sites in Rat Forebrain

Abstract: The ontogeny of binding sites for [³H] spiperone was studied in time-pregnant rats. Binding of [³H]spiperone to fresh homogenates of pre- and postnatal rat forebrain was characterized by Scatchard analysis and competition experiments with a number of dopaminergic and serotonergic agonists and antagonists and additional substances. A convenient discrimination of three high-affinity sites, i.e., the dopaminergic D₂, serotonergic S₂, and spirodecanone (Sd) sites, was obtained with l-(–)sulpiride and cis-flupenthixol. The analgesic R5573 was found not to be specific for the Sd site but to interact with all three sites. The three binding sites became detectable in sequential order. S₂ and D₂ binding sites were first found at embryonic days 15.75 and 17.75, respectively. The Sd site did not appear before postnatal day 8. All three binding sites reached adult values at approximately postnatal day 30. During the prenatal period, the increase in the number of D₂ binding sites paralleled the rise in forebrain dopamine concentrations. The kinetics of D₂ and S₂ sites were the same at embryonic day 19.75 and postnatal day 30. These observations provide evidence for the presence of the receptor substrate for actions of neuroleptics on dopaminergic and serotonergic systems during fetal life.     

Synthesis and characterization of selective dopamine D2 receptor antagonists. 2. Azaindole,benzofuran, and benzothiophene analogs of L-741,626

A series of indole, 7-azaindole, benzofuran, and benzothiophene compounds have been prepared and evaluated for affinity at D_2-like dopamine receptors. These compounds share structural elements with the classical D_2-like dopamine receptor antagonists haloperidol, N-methylspiperone and benperidol. Two new compounds, 4-(4-iodophenyl)-1-((4-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol (6) and 4-(4-iodophenyl)-1-((5-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol (7), were found to have high affinity to and selectivity for D₂ versus D₃ receptors. Changing the aromatic ring system from an indole to other heteroaromatic ring systems reduced the D₂ binding affinity and the D₂ versus D₃ selectivity.     

Design and synthesis of D1 agonist/D2 antagonist for treatment of schizophrenia

A series of tetrahydroisoquinolines were designed, synthesized and evaluated as the first non-natural product type of compounds with dual D₁ receptor (D₁R) agonism and D₂ receptor (D₂R) antagonism properties for treatment of schizophrenia. The initial SAR of the series was explored. The lead in the series, 3g, exhibited high affinity and good potency. Compound 3g displayed 95% of D₁R occupancy (10 mg/kg, sc) and 75% of D₂R occupancy (10 mg/kg, sc) in the striatum of male CD-1 mice. The series exhibited unique pharmacology and merit as tool compounds for target validation and future optimizations.     

Beta-adrenergic receptor in the brain: comparison of 3H-dihydroalprenolol binding sites and a beta-adrenergic receptor regulating adenylyl cyclase activity in cell free homogenates.

The properties of specific ³H-dihydroalprenolol binding sites resemble the properties of the beta-receptor regulating hormone-sensitive adenylyl cyclase activity in an homogenate of rabbit cerebellum. The rabbit cerebellum has 5 to 6 pmole per gm (wet weight) of high affinity (K_D=1.3 nM) specific binding sites for ³H-dihydroalprenolol. the interaction of several beta-adrenergic agonists and antagonists with the specific binding sites is rapid, reversible, and demonstrates stereospecificity which parallels the properties of the beta receptor. Beta-adrenergic agonists show a similar potency as agonists upon adenylyl cyclase activity and as inhibitors of ³H-dihydroalprenolol binding: i.e. l-isoproterenol > l-epinephrine > l-norepinephrine (suggesting a beta₂ adrenergic receptor). The binding affinities of several beta-adrenergic agonists and antagonists for the specific binding sites approximate the affinities of these compounds for the stimulation of adenylyl cyclase. Thus, the ³H-dihydroalprenolol binding sites have properties similar to the beta-adrenergic receptor regulating adenylyl cyclase activity in a rabbit cerebellar homogenate.     

[3-Azabicyclo[3.1.0]hex-1-yl]phenyl-benzenesulfonamides as selective dopamine D3 antagonists

A new class of azabicyclo[3.1.0]benzenesulfonamides is presented as selective dopamine D₃ antagonists together with SAR and selectivity data.     

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: Dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors—Part II

8-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones have been identified as highly potent 5-HT_1A/B/D receptor antagonists with and without additional SerT activity and a high degree of selectivity over hERG potassium channels. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

The identification of a selective dopamine D2 partial agonist,D3 antagonist displaying high levels of brain exposure

The identification of a highly selective D₂ partial agonist, D₃ antagonist tool molecule which demonstrates high levels of brain exposure and selectivity against an extensive range of dopamine, serotonin, adrenergic, histamine, and muscarinic receptors is described.     

Investigation of orexin-2 selective receptor antagonists: Structural modifications resulting in dual orexin receptor antagonists

In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX₂R subtype and culminating in the discovery of 23, a highly potent, OX₂R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX₁R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.     

Synthesis and biological evaluation of bivalent cannabinoid receptor ligands based on hCB2R selective benzimidazoles reveal unexpected intrinsic properties

The design of bivalent ligands targeting G protein-coupled receptors (GPCRs) often leads to the development of new, highly selective and potent compounds. To date, no bivalent ligands for the human cannabinoid receptor type 2 (hCB₂R) of the endocannabinoid system (ECS) are described. Therefore, two sets of homobivalent ligands containing as parent structure the hCB₂R selective agonist 13a and coupled at different attachment positions were synthesized. Changes of the parent structure at these positions have a crucial effect on the potency and efficacy of the ligands. However, we discovered that bivalency has an influence on the effect at both cannabinoid receptors. Moreover, we found out that the spacer length and the attachment position altered the efficacy of the bivalent ligands at the receptors by turning agonists into antagonists and inverse agonists.