Robert R. Luedtke

RGS9 modulates dopamine signaling in the basal ganglia.

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. We examined a role for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduced locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice showed heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerated the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increased RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness.

Molecular determinants of selectivity and efficacy at the dopamine D3 receptor.

The dopamine D3 receptor (D3R) has been implicated in substance abuse and other neuropsychiatric disorders. The high sequence homology between the D3R and D2R, especially within the orthosteric binding site (OBS) that binds dopamine, has made the development of D3R-selective compounds challenging. Here, we deconstruct into pharmacophoric elements a series of D3R-selective substituted-4-phenylpiperazine compounds and use computational simulations and binding and activation studies to dissect the structural bases for D3R selectivity and efficacy. We find that selectivity arises from divergent interactions within a second binding pocket (SBP) separate from the OBS, whereas efficacy depends on the binding mode in the OBS. Our findings reveal structural features of the receptor that are critical to selectivity and efficacy that can be used to design highly D3R-selective ligands with targeted efficacies. These findings are generalizable to other GPCRs in which the SBP can be targeted by bitopic or allosteric ligands.

Progress in developing D3 dopamine receptor ligands as potential therapeutic agents for neurological and neuropsychiatric disorders.

The dopamine D3 receptor has been the subject of a tremendous amount of research since its discovery in 1990. A previous review of this subject [3] described the advances in molecular biology and neuroanatomical localization of the D3 receptor, with a special emphasis on schizophrenia. In the current review, we attempt to describe recent advances in the biochemistry and pharmacology of the D3 receptor from the molecular to the behavioral level. Evidence linking an alteration in D3 receptor function as playing an important role in the etiology of a variety of CNS disorders, including schizophrenia, Parkinsons Disease, and substance abuse is also provided. Also discussed are the recent developments in attempting to map the ligand-binding domains of the D2 and D3 receptors. A. survey of the literature, including a description of the medicinal chemistry approaches toward developing D3-selective ligands, is also presented in this review.

N-(4-(4-(2,3-Dichloro- or 2-methoxyphenyl)piperazin-1-yl)-butyl)-heterobiarylcarboxamides with Functionalized Linking Chains as High Affinity and Enantioselective D3 Receptor Antagonists

In the present report, the D3 receptor pharmacophore is modified in the 2,3-diCl- and 2-OCH(3)-phenylpiperazine class of compounds with the goal to improve D3 receptor affinity and selectivity. This extension of structure-activity relationships (SAR) has resulted in the identification of the first enantioselective D3 antagonists (R- and S-22) to be reported, wherein enantioselectivity is more pronounced at D3 than at D2, and that a binding region on the second extracellular loop (E2) may play a role in both enantioselectivity and D3 receptor selectivity. Moreover, we have discovered some of the most D3-selective compounds reported to date that show high affinity (K(i) = 1 nM) for D3 and approximately 400-fold selectivity over the D2 receptor subtype. Several of these analogues showed exquisite selectivity for D3 receptors over >60 other receptors, further underscoring their value as in vivo research tools. These lead compounds also have appropriate physical characteristics for in vivo exploration and therefore will be useful in determining how intrinsic activity at D3 receptors tested in vitro is related to behaviors in animal models of addiction and other neuropsychiatric disorders.

N-{4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl, butenyl and butynyl}arylcarboxamides as novel dopamine D3 receptor antagonists

The dopamine D(3) receptor subtype has been targeted as a potential neurochemical modulator of the behavioral actions of psychomotor stimulants, such as cocaine. Previous synthetic studies provided structural requirements for high affinity binding to D(3) receptors which included a 2,3-dichloro-phenylpiperazine linked to an arylamido function via a butyl chain. To reduce lipophilicity of these agents and further investigate optimal conformation, a second series of 15 novel ligands was designed that included heteroaromatic substitution and unsaturated alkyl linkers. These compounds were synthesized and evaluated for binding at rat D(3) and D(2) receptors stably expressed in Sf9 cells. D(3) binding affinities ranged from K(i)=0.6-1080 nM, with a broad range of D(3)/D(2) selectivities (2-97). The discovery of potent, selective and bioavailable D(3) receptor ligands will provide essential molecular probes to elucidate the role D(3) receptors play in the psychomotor stimulant and reinforcing effects of cocaine.

Immunoblot and immunohistochemical comparison of murine monoclonal antibodies specific for the rat D1a and D1b dopamine receptor subtypes

The two D1-like dopamine receptor subtypes, D1a and D1b, are structurally similar and pharmacologically indistinguishable using currently available ligands. To differentiate between the D1-like dopamine receptor subtypes, murine monoclonal antibodies to the rat Dla and the rat D1b dopamine receptor have been prepared. Rat D1-like and D2-like dopamine receptors expressed in Sf9 cells were used to verify the immunospecificity of the monoclonal anti-(D1a dopamine receptor) and anti-(D1b dopamine receptor) antibodies using immunoblot and immunohistochemical techniques. These two antibodies were used to compare the temporal dynamics of D1-like dopamine receptors expressed in Sf9 cells following infection with recombinant baculovirus and to monitor the partial purification of detergent solubilized receptors following ion exchange chromatography. Immunoreactivity of the anti-(D1a receptor) antibody was observed in the striatum and cortical regions of the rat brain using immunoblot techniques. No reactivity on immunoblots was observed for the anti-(D1b receptor) antibody using rat brain tissue, probably due to the low levels of receptor expression. For immunohistochemical studies using rat brain slices, the anti-(D1a receptor) antibody heterogeneously labeled cells and punctate processes within the striatal neuropil while labeling in the adjacent cerebral cortex was weak. Anti-(D1b receptor) antibody immunoreactivity was weak in the .striatum and generally limited to sparse perikarya in the dorsal region. However, immunoreactivity was observed in numerous cells within the vertical and horizontal limbs of the diagonal band and in the ventral pallidum. Immunoreactivity of the anti-(D1b receptor) antibody was also observed in layer V pyramidal neurons of the frontal sensorimotor cortex.

Quantitative Changes in G α olf Protein Levels, but not D1 Receptor, Alter Specifically Acute Responses to Psychostimulants

Striatal dopamine D1 receptors (D1R) are coupled to adenylyl cyclase through Gαolf. Although this pathway is involved in important brain functions, the consequences of quantitative alterations of its components are not known. We explored the biochemical and behavioral responses to cocaine and D-amphetamine (D-amph) in mice with heterozygous mutations of genes encoding D1R and Gαolf (Drd1a+/− and Gnal+/−), which express decreased levels of the corresponding proteins in the striatum. Dopamine-stimulated cAMP production in vitro and phosphorylation of AMPA receptor GluR1 subunit in response to D-amph in vivo were decreased in Gnal+/−, but not Drd1a+/− mice. Acute locomotor responses to D1 agonist SKF81259, D-amph and cocaine were altered in Gnal+/− mice, and not in Drd1a+/− mice. This haploinsufficiency showed that Gαolf but not D1R protein levels are limiting for D1R-mediated biochemical and behavioral responses. Gnal+/− mice developed pronounced locomotor sensitization and conditioned locomotor responses after repeated injections of D-amph (2 mg/kg) or cocaine (20 mg/kg). They also developed normal D-amph-conditioned place preference. The D1R/cAMP pathway remained blunted in repeatedly treated Gnal+/− mice. In contrast, D-amph-induced ERK activation was normal in the striatum of these mice, possibly accounting for the normal development of long-lasting behavioral responses to psychostimulants. Our results clearly dissociate biochemical mechanisms involved in acute and delayed behavioral effects of psychostimulants. They identify striatal levels of Gαolf as a key factor for acute responses to psychostimulants and suggest that quantitative alterations of its expression may alter specific responses to drugs of abuse, or possibly other behavioral responses linked to dopamine function.

N-(3-fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-1-yl)butyl)arylcarboxamides as selective dopamine D3 receptor ligands: critical role of the carboxamide linker for D3 receptor selectivity.

N-(3-fluoro-4-(4-(2,3-dichloro- or 2-methoxyphenyl)piperazine-1-yl)butyl)arylcarboxamides were prepared and evaluated for binding and function at dopamine D3 receptors (D3Rs) and dopamine D2 receptors (D2Rs). In this series, we discovered some of the most D3R selective compounds reported to date (e.g., 8d and 8j, >1000-fold D3R-selective over D2R). In addition, chimeric receptor studies further identified the second extracellular (E2) loop as an important contributor to D3R binding selectivity. Further, compounds lacking the carbonyl group in the amide linker were synthesized, and while these amine-linked analogues bound with similar affinities to the amides at D2R, this modification dramatically reduced binding affinities at D3R by >100-fold (e.g., D3R K(i) for 15b = 393 vs for 8j = 2.6 nM), resulting in compounds with significantly reduced D3R selectivity. This study supports a pivotal role for the D3R E2 loop and the carbonyl group in the 4-phenylpiperazine class of compounds and further reveals a point of separation between structure-activity relationships at D3R and D2R.

Evaluation of D2 and D3 dopamine receptor selective compounds on l-dopa-dependent abnormal involuntary movements in rats

A panel of novel D2 and D3 dopamine receptor selective antagonists, partial agonists and full agonists have been evaluated for the ability to attenuate L-dopa-associated abnormal involuntary movements (AIMs) in 6-hydroxydopamine (6-OHDA) unilaterally lesioned male Sprague Dawley rats, which is an animal model of L-dopa-induced dyskinesia (LID). LID is often observed in patients with Parkinsons Disease following chronic treatment with L-dopa. The intrinsic activity of these dopaminergic compounds was determined using a forskolin-dependent adenylyl cyclase inhibition assay with transfected HEK 293 cells expressing either the human D2Long or D3 dopamine receptor subtype. For the initial experiments the 5-HT1A receptor selective partial agonist buspirone was used to verify our ability to quantitate changes in total AIMs and AIMs minus locomotor scores. Two D2 dopamine receptor selective antagonists, SV 156 and SV 293, were evaluated and found to minimally attenuate AIM scores in these animals. Four members of our WC series of D3 dopamine receptor selective compounds of varying intrinsic activity at the D3 dopamine receptor subtype, WC 10, WC 21, WC 26 and WC 44, were also evaluated and found to attenuate AIM scores in a dose dependent manner. The in vivo efficacy of the compounds increased when they were administered simultaneously with L-dopa, as compared to when the compounds were administered 60 min prior to the L-dopa/benserazide. It was also found that the D3 receptor antagonist WC 10 could inhibit the involuntary movements after they had achieved maximum intensity. Unlike the D1-like dopamine receptor selective agonist SKF 81297 and the D2-like dopamine receptor agonist bromocriptine which can precipitate abnormal involuntary movements in these unilaterally lesioned animals, abnormal involuntary movements were not observed after administration of our D3 receptor selective agonist WC 44. In addition, we evaluated the effect of these four D3 dopamine receptor selective compounds for their effect on a) spontaneous locomotion and b) coordination and agility using a rotarod apparatus. We also used a cylinder test to assess the effect of L-dopa on spontaneous and independent use of each of the rats forelimbs in the presence or absence of test compound. The results of these studies suggest that substituted phenylpiperazine D3 dopamine receptor selective compounds are potential pharmacotherapeutic agents for the treatment of L-dopa-associated dyskinesia in patients with Parkinsons Disease.

The Tetrahydroisoquinoline Derivative SB269,652 Is an Allosteric Antagonist at Dopamine D3 and D2 Receptors

In view of the therapeutic importance of dopamine D3 and D2 receptors, there remains considerable interest in novel ligands. Herein, we show that the tetrahydroisoquinoline 1H-indole-2-carboxylic acid {4-[2-(cyano-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-cyclohexyl}-amide (SB269,652) behaves as an atypical, allosteric antagonist at D3 and D2 receptors. Accordingly, SB269,652 potently (low nanomolar range) abolished specific binding of [3H]nemanopride and [3H]spiperone to Chinese hamster ovary-transfected D3 receptors when radioligands were used at 0.2 and 0.5 nM, respectively. However, even at high concentrations (5 μM), SB269,652 only submaximally inhibited the specific binding of these radioligands when they were employed at 10-fold higher concentrations. By analogy, although SB269,652 potently blocked D3 receptor-mediated activation of Gαi3 and phosphorylation of extracellular-signal-regulated kinase (ERK)1/2, when concentrations of dopamine were increased by 10-fold, from 1 μM to 10 μM, SB269,652 only submaximally inhibited dopamine-induced stimulation of Gαi3. SB269,652 (up to 10 μM) only weakly and partially (by approximately 20–30%) inhibited radioligand binding to D2 receptors. Likewise, SB269,652 only submaximally suppressed D2 receptor-mediated stimulation of Gαi3 and Gαqi5 (detected with the aequorin assay) and phosphorylation of ERK1/2 and Akt. Furthermore, SB269,652 only partially (35%) inhibited the dopamine-induced recruitment of β-arrestin2 to D2 receptors. Finally, Schild analysis using Gαi3 assays, and studies of radioligand association and dissociation kinetics, supported allosteric actions of SB269,652 at D3 and D2 receptors.