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Biased signaling agonist of Dopamine D3 receptor induces receptor internalization independent of β-Arrestin recruitment

Xu, Wei; Reith, Maarten E A; Chen, Lee-Yuan Liu; Kortagere, Sandhya
Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a significant step in receptor kinetics and is known to be involved in receptor down-regulation. However, the dopamine D3 receptor (D3R) has been an exception wherein agonist induces D3Rs to undergo desensitization followed by pharmacological sequestration - which is defined as the sequestration of cell surface receptors into a more hydrophobic fraction within the plasma membrane without undergoing the process of receptor internalization. Pharmacological sequestration renders the receptor in an inactive state on the membrane. In our previous study we demonstrated that a novel class of D3R agonists exemplified by SK608 have biased signaling properties via the G-protein dependent pathway and do not induce D3R desensitization. In this study, using radioligand binding assay, immunoblot or immunocytochemistry methods, we observed that SK608 induced internalization of human D3R stably expressed in CHO, HEK and SH-SY5Y cells which are derived from neuroblastoma cells, suggesting that it is not a cell-type specific event. Further, we have evaluated the potential mechanism of D3R internalization induced by these biased signaling agonists. SK608-induced D3R internalization was time- and concentration-dependent. In comparison, dopamine induced D3R upregulation and pharmacological sequestration in the same assays. GRK2 and clathrin/dynamin I/II are the key molecular players in the SK608-induced D3R internalization process, while β-arrestin 1/2 and GRK-interacting protein 1(GIT1) are not involved. These results suggest that SK608-promoted D3R internalization is similar to the type II internalization observed among peptide binding GPCRs.
PMID: 30844536
ISSN: 1096-1186
CID: 3723412

Design, Synthesis and Pharmacological Characterization of Carbazole Based Dopamine Agonists as Potential Symptomatic and Neuroprotective Therapeutic Agents for Parkinson's Disease

Elmabruk, Asma; Das, Banibrata; Yedlapudi, Deepthi; Xu, Liping; Antonio, Tamara; Reith, Maarten E A; Dutta, Aloke K
We have developed a series of carbazole-derived compounds based on our hybrid D2/D3 agonist template to design multifunctional compounds for the symptomatic and disease-modifying treatment of Parkinson's disease (PD). The lead molecules (-)-11b, (-)-15a and (-)-15c exhibited high affinity for both D2 and D3 receptors where as in GTPγS functional assay, the compounds showed potent agonist activity at both D2 and D3 receptors (EC50 (GTPγS); D2 = 48.7 nM, D3 = 0.96 nM for 11b, D2 = 0.87 nM, D3 = 0.23 nM for 15a and D2 = 2.29 nM, D3 = 0.22 nM for 15c). In PD animal model study, the test compounds exhibited potent in vivo activity in reversing hypolocomotion in reserpinized rats with a long duration of action compared to the reference drug. In a cellular antioxidant assay, compounds (-)-11b, (-)-15a and (-)-15c exhibited potent activity in reducing oxidative stress induced by neurotoxin 6-hydroxydopamine (6-OHDA). Also, in a cell-based PD neuroprotection model, these lead compounds significantly increased cell survival from toxicity of 6-OHDA, thereby, producing neuroprotection effect. Additionally, compounds D636 and D653 inhibited aggregation and reduced toxicity of recombinant alpha synuclein protein in a cell based in vitro assays. These observations suggest that the lead carbazole-based dopamine agonists may be promising multifunctional molecules for a viable symptomatic and disease modifying therapy of PD and should be further investigated.
PMID: 30301349
ISSN: 1948-7193
CID: 3353352

Selective activation of Dopamine D3 receptors and norepinephrine transporter blockade enhance sustained attention

Marshall, Courtney A; Brodnik, Zachary D; Mortensen, Ole V; Reith, Maarten E A; Shumsky, Jed S; Waterhouse, Barry D; España, Rodrigo A; Kortagere, Sandhya
Catecholamine transmitters dopamine (DA) and norepinephrine (NE) regulate prefrontal cortical (PFC) circuit activity and PFC-mediated executive functions. Accordingly, pharmacological agents that influence catecholamine neurotransmission exert prominent effects on cognition. Many such agents are used clinically to treat attention disorders. For example, methylphenidate blocks DA and NE reuptake and is the leading choice for attention deficit hyperactivity disorder (ADHD) treatment. Recently, we have designed SK609 - a selective small molecule agonist of the DA D3 receptor (D3R). In this study, we further characterized SK609's ability to selectively inhibit the reuptake of NE by NE transporters (NET). Our results indicate SK609 selectively inhibits NET with a Ki value of ∼500 nM and behaves as a NET substrate. Systemic dosing of SK609 (4 mg/kg; i.p.) in naïve rats produced a 300% and 160% increase in NE and DA, respectively, in the PFC as measured by microdialysis. Based on these neurochemical results, SK609 was tested in a PFC-dependent, visually-guided sustained attention task in naïve rats. SK609 improved performance in a dose-dependent manner with a classical inverted-U dose response function with a peak effect at 4 mg/kg. SK609's peak effect was blocked by a pre-treatment with either the D2/D3R antagonist raclopride (0.05 mg/kg; i.p) or the alpha-1 adrenergic receptor antagonist prazosin (0.25 mg/kg; i.p), confirming a role for both DA and NE in promoting sustained attention. Additionally, SK609 improved sustained attention more prominently among low-performing animals. Doses of SK609 (2, 4, and 8 mg/kg) associated with cognitive enhancement did not produce an increase in spontaneous locomotor activity, suggesting a lack of side effects mediated by DA transporter (DAT) activity. These results demonstrate that the novel catecholaminergic modulator SK609 has the potential to treat sustained attention deficits without affecting DAT activity, distinguishing it from amphetamines and methylphenidate.
PMID: 30633928
ISSN: 1873-7064
CID: 3580032

A Novel Orally Active Triple Reuptake Inhibitor for the Treatment of Post-Traumatic Stress Disorder (PTSD): D-578 Attenuates Abnormal Fear Behavior in a Rodent Model of Traumatic Stress [Meeting Abstract]

Lisieski, Michael; Harutyunyan, Arman; Das, Banibrata; Liberzon, Israel; Bymaster, Frank; Hsu, Timothy; Reith, Maarten; Perrine, Shane.; Dutta, Aloke
ISSN: 0006-3223
CID: 3974062

Tamoxifen Directly Interacts with the Dopamine Transporter

Mikelman, Sarah R; Guptaroy, Bipasha; Schmitt, Kyle C; Jones, Kymry T; Zhen, Juan; Reith, Maarten E A; Gnegy, Margaret E
The selective estrogen receptor modulator tamoxifen increases extracellular dopamine in vivo and act as a neuroprotectant in models of dopamine neurotoxicity. We investigated the effect of tamoxifen on dopamine transporter (DAT)-mediated dopamine uptake, dopamine efflux, and [3H]WIN 35,428 binding in rat striatal tissue. Tamoxifen dose dependently blocks dopamine uptake (54% reduction at 10 μM) and amphetamine-stimulated efflux (59% reduction at 10 μM) in synaptosomes. It also produces a small but significant reduction in [3H] WIN35,428 binding in striatal membranes, indicating a weak interaction with the substrate binding site in the DAT. Biotinylation and cysteine accessibility studies indicate that tamoxifen stabilizes the outward-facing conformation of the dopamine transporter in a cocaine-like manner and does not affect surface expression of the DAT. Additional studies with mutant DAT constructs D476A and I159A suggest a direct interaction between tamoxifen and a secondary substrate binding site of the transporter. Locomotor studies reveal that tamoxifen attenuates amphetamine-stimulated hyperactivity in rats, but has no depressant or stimulant activity in the absence of amphetamine. These results suggest a complex mechanism of action for tamoxifen as a regulator of the DAT. Due to its effectiveness against amphetamine actions and its CNS permeant activity, the tamoxifen structure represents an excellent starting point for a structure-based drug-design program to develop a pharmacological therapeutic for psychostimulant abuse.
PMID: 30108161
ISSN: 1521-0103
CID: 3254642

Latch and trigger role for R445 in DAT transport explains molecular basis of DTDS

Reith, Maarten E A; Jones, Kymry T; Zhen, Juan; Topiol, Sid
A recent study reports on five different mutations as sources of dopamine transporter (DAT) deficiency syndrome (DTDS). One of these mutations, R445C, is believed to be located on the intracellular side of DAT distal to the primary (S1) or secondary (S2) sites to which substrate binding is understood to occur. Thus, the molecular mechanism by which the R445C mutation results in DAT transport deficiency has eluded explanation. However, the recently reported X-ray structures of the endogenous amine transporters for dDAT and hSERT revealed the presence of a putative salt bridge between R445 and E428 suggesting a possible mechanism. To evaluate whether the R445C effect is a result of a salt bridge interaction, the mutants R445E, E428R, and the double mutant E428R/R445E were generated. The single mutants R445E and E428R displayed loss of binding and transport properties of the substrate [3H]DA and inhibitor [3H]CFT at the cell surface while the double mutant E428R/R445E, although nonfunctional, restored [3H]DA and [3H]CFT binding affinity to that of WT. Structure based analyses of these results led to a model wherein R445 plays a dual role in normal DAT function. R445 acts as a component of a latch in its formation of a salt bridge with E428 which holds the primary substrate binding site (S1) in place and helps enforce the inward closed protein state. When this salt bridge is broken, R445 acts as a trigger which disrupts a local polar network and leads to the release of the N-terminus from its position inducing the inward closed state to one allowing the inward open state. In this manner, both the loss of binding and transport properties of the R445C variant are explained.
PMID: 29258773
ISSN: 1464-3405
CID: 2894042

Functional properties of dopamine transporter oligomers after copper linking

Zhen, Juan; Reith, Maarten E A
Although it is universally accepted that dopamine transporters (DATs) exist in monomers, dimers and tetramers (i.e. dimers of dimers), it is not known whether the oligomeric organization of DAT is a prerequisite for its ability to take up dopamine (DA), or whether each DAT protomer-the subunit of quaternary structure- functions independently in terms of DA translocation. In the present work copper phenanthroline (CuP) was used to selectively target surface DAT: increasing concentrations of CuP gradually cross-linked natural DAT dimers in LLC-PK1 cells stably expressing hDAT and thereby reduced DA uptake functionality until all surface DATs were inactivated. DATs that were not cross-linked by CuP showed normal DA uptake with DA Km at ~ 0.5 muM and DA efflux with basal and amphetamine-induced DA efflux as much as control values. The cocaine analog 2beta-carbomethoxy-3beta-[4-fluorophenyl]-tropane (CFT) was capable to bind to copper-cross-linked DATs, albeit with an affinity more than 5 fold decreased (Kd of CFT =109 nM after crosslinking vs 19 nM before). A kinetic analysis is offered describing the changing amounts of dimers and monomers with increasing [CuP], allowing the estimation of dimer functional activity compared with a DAT monomer. Consonant with previous conclusions for SERT and NET that only one protomer of an oligomer is active at the time, the present data indicated -a functional activity of the DAT dimer of 0.74 relative to a monomer.
PMID: 29168892
ISSN: 1471-4159
CID: 2792172

Dopamine Transporter Phosphorylation Site Threonine 53 is Stimulated by Amphetamines and Regulates Dopamine Transport, Efflux, and Cocaine Analog Binding

Challasivakanaka, Sathya; Zhen, Juan; Smith, Margaret E; Reith, Maarten E A; Foster, James D; Vaughan, Roxanne A
The dopamine transporter (DAT) controls the spatial and temporal dynamics of dopamine neurotransmission through reuptake of extracellular transmitter and is a target for addictive compounds such as cocaine, amphetamine (AMPH), and methamphetamine (METH). Reuptake is regulated by kinase pathways and drug exposure, allowing for fine-tuning of clearance in response to specific conditions, and here we examine the impact of transporter ligands on DAT residue Thr53, a proline-directed phosphorylation site previously implicated in AMPH-stimulated efflux mechanisms. Our findings show that Thr53 phosphorylation is stimulated in a transporter-dependent manner by AMPH and METH in model cells and rat striatal synaptosomes, and in striatum of rats given subcutaneous injection of METH. Rotating disc electrode voltammetry revealed that initial rates of uptake and AMPH-induced efflux were elevated in phosphorylation-null T53A DAT relative to WT and charge-substituted T53D DATs, consistent with functions related to charge or polarity. These effects occurred without alterations of surface transporter levels, and mutants also showed reduced cocaine analog binding affinity that was not rescued by Zn2+ Together these findings support a role for Thr53 phosphorylation in regulation of transporter kinetic properties that could impact DAT responses to amphetamines and cocaine.
PMID: 28939767
ISSN: 1083-351x
CID: 2708492

A Novel Iron(II) Preferring Dopamine Agonist Chelator as Potential Symptomatic and Neuroprotective Therapeutic Agent for Parkinson's Disease

Das, Banibrata; Kandegedara, Ashoka; Xu, Liping; Antonio, Tamara; Stemmler, Timothy; Reith, Maarten E A; Dutta, Aloke K
Parkinson's disease (PD) is a progressive neurodegenerative disorder, and development of disease-modifying treatment is still an unmet medical need. Considering the implication of free iron(II) in PD, we report here the design and characterization of a novel hybrid iron chelator, (-)-12 (D-607) as a multitarget-directed ligand against PD. Binding and functional assays at dopamine D2/D3 receptors indicate potent agonist activity of (-)-12. The molecule displayed an efficient preferential iron(II) chelation properties along with potent in vivo activity in a reserpinized PD animal model. The compound also rescued PC12 cells from toxicity induced by iron delivered intracellularly in a dose-dependent manner. However, Fe3+ selective dopamine agonist 1 and a well-known antiparkinsonian drug pramipexole produced little to no neuroprotection effect under the same experimental condition. These observations strongly suggest that (-)-12 should be a promising multifunctional lead molecule for a viable symptomatic and disease modifying therapy of PD.
PMID: 28106982
ISSN: 1948-7193
CID: 2560122

Functional Characterization of a Novel Series of Biased Signaling Dopamine D3 Receptor Agonists

Xu, Wei; Wang, Xiaozhao; Tocker, Aaron M; Huang, Peng; Reith, Maarten E A; Liu-Chen, Lee-Yuan; Smith, Amos B 3rd; Kortagere, Sandhya
Dopamine receptors play an integral role in controlling brain physiology. Importantly, subtype selective agonists and antagonists of dopamine receptors with biased signaling properties have been successful in treating psychiatric disorders with a low incidence of side effects. To this end, we recently designed and developed SK609, a dopamine D3 receptor (D3R) selective agonist that has atypical signaling properties. SK609 has shown efficacy in reversing akinesia and reducing L-dopa-induced dyskinesia in a hemiparkinsonian rats. In the current study, we demonstrate that SK609 has high selectivity for D3R with no binding affinity on D2R high- or low-affinity state when tested at a concentration of 10 muM. In addition, SK609 and its analogues do not induce desensitization of D3R as determined by repeated agonist treatment response in phosphorylation of ERK1/2 functional assay. Most significantly, SK609 and its analogues preferentially signal through the G-protein-dependent pathway and do not recruit beta-arrestin-2, suggesting a functional bias toward the G-protein-dependent pathway. Structure-activity relationship (SAR) studies using analogues of SK609 demonstrate that the molecules bind at the orthosteric site by maintaining the conserved salt bridge interactions with aspartate 110 on transmembrane 3 and aryl interactions with histidine 349 on transmembrane 6, in addition to several hydrophobic interactions with residues from transmembranes 5 and 6. The compounds follow a strict SAR with reference to the three pharmacophore elements: substituted phenyl ring, length of the linker connecting phenyl ring and amine group, and orientation and hydrophobic branching groups at the amine among SK609 analogues for efficacy and functional selectivity. These features of SK609 and the analogues suggest that biased signaling is an inherent property of this series of molecules.
PMID: 27801563
ISSN: 1948-7193
CID: 2488292