The Lignan (−)-Hinokinin Displays Modulatory Effects on Human

Article pubs.acs.org/jnp

The Lignan (−)-Hinokinin Displays Modulatory Effects on Human Monoamine and GABA Transporter Activities Julie Marie V. Timple,†,§ Lizandra Guidi Magalhaẽ s,‡ Karen Cristina Souza Rezende,‡ Ana Carolina Pereira,‡ Wilson Roberto Cunha,‡ Márcio Luis Andrade e Silva,‡ Ole Valente Mortensen,† and Andréia C. K. Fontana*,†,§ †

Department of Pharmacology and Physiology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102, United States ‡ Núcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, CEP 14404-600, Franca, SP, Brazil S Supporting Information *

ABSTRACT: The neurotransmitter transporters of the SLC6 family play critical roles in the regulation of neurotransmission and are the primary targets of therapeutic agents used to treat clinical disorders involving compromised neurotransmitter signaling. The dopamine and norepinephrine transporters have been implicated in clinical disorders such as attention deficit hyperactivity disorder (ADHD) and substance abuse. The GABA transporters (GATs) serve as a target for anxiolytic, antidepressant, and antiepileptic therapies. In this work, the interaction with neurotransmitter transporters was characterized for a derivative of the lignan (−)-cubebin (1), namely, (−)-hinokinin (2). Using in vitro pharmacological assays, 2 selectively inhibited the human dopamine and norepinephrine transporters, in a noncompetitive manner possibly mediated by binding to a novel site within the transporters, and displayed low affinity for the serotonin transporter. Compound 2 also specifically inhibited the GAT-1 GABA transporter subtype. Compound 2 is not a substrate of the carriers as it had no effect on the efflux of either of the neurotransmitters investigated. This compound is inactive toward glutamate and glycine transporters. These results suggest that 2 may serve as a tool to develop new therapeutic drugs for ADHD and anxiety that target the DAT, NET, and GAT-1 transporters.

N

cleft, thereby terminating dopaminergic and norepinergic signaling.8 Currently, about 5 million children ranging from ages 3 to 17 are diagnosed with attention deficit hyperactivity disorder (ADHD), a neurobehavioral disorder that has the DAT and NET as therapeutic targets for treatment.9 Disruptions in the extracellular availability of dopamine and norepinephrine have been implicated in ADHD.10 This deficiency can be overcome, through inhibition of DAT and NET, leading to a subsequent increase in substrate availability and reduction of symptoms related to ADHD.11−13 The current therapies for this psychiatric condition consist primarily of the DAT and NET substrate amphetamine (Adderall) and the DAT and NET inhibitor and benzylpiperidine derivative methylphenidate (Ritalin).14,15 Amphetamines act as carrier substrates by competing with the endogenous substrates and can also elicit efflux of dopamine or norepinephrine into the synaptic cleft from the presynaptic terminal.5,16−18 Additionally, atomoxetine (Strattera) is a selective NET inhibitor that is used in the treatment of ADHD.19 NET has also been implicated in various

eurotransmitter transporters play critical roles in the regulation of neurotransmission that modulates physiological responses involving homeostasis, stress response, behavior, motivation, and reward.1,2 Under normal physiological processes, neurotransmitters are stored in vesicles in the presynaptic neuron and released into the synaptic cleft to activate or inhibit postsynaptic receptors. The downstream signaling of these neurotransmitters is terminated by reuptake of the substrate through its respective transporter.3 Alteration in the regulation of extracellular neurotransmitter levels can lead to various neurological and mental health conditions, and subsequently, neurotransmitter transporters have become a target of interest for therapies altering neurotransmitter clearance.4,5 Dopamine is involved in neurological processes of rewarddriven learning and plays an important role in the normal functions of attention, mood, memory, and sleep.6 Norepinephrine plays a role in learning, memory, attention, and pain perception.7 The classical biogenic monoamine transporters, the human dopamine transporter (hDAT) and norepinephrine transporters (hNET), are critical for the regulation of dopamine and norepinephrine signaling, as they are responsible for the clearance of the respective neurotransmitters from the synaptic © 2013 American Chemical Society and American Society of Pharmacognosy

Received: June 6, 2013 Published: October 10, 2013 1889

dx.doi.org/10.1021/np400452n | J. Nat. Prod. 2013, 76, 1889−1895

Journal of Natural Products

Article

Transport. In the first series of assays performed, the effects of 2 were evaluated in several transporter-mediated assays. Dose responses of 2 in hDAT, hSERT, hNET, and GAT-1 transport assays are shown in Figure 1A−D, respectively. Compound 2 inhibited dopamine transport in a dose-dependent manner with an IC50 value of 8.35 ± 0.2 μM (Figure 1A). On the other hand, 2 showed a lower affinity for inhibiting serotonin transport, with an IC50 value of 175 ± 3.7 μM (Figure 1B). Norepinephrine transport assays resulted in an IC50 value of 8.76 ± 0.4 μM for compound 2 (Figure 1C), indicating that this compound has high selectivity for hNET and hDAT over hSERT (Figure 1C and Table 1). GAT-1 transport assays revealed an IC50 value of 20.6 ± 0.4 μM (Figure 1D). Differently from DAT and SERT, for both NET and GAT-1, a plateau was observed in the inhibition, as 2 did not completely inhibit these two transporters. It is particularly interesting to note that the potency of (−)-hinokinin in inhibiting hDAT and hNET is similar, but this compound did not completely inhibit hNET activity and fully inhibited DAT at the highest concentrations. The mechanism of inhibition by 2 between the two transporters must therefore be subtly different. The finding also suggests that when the binding of 2 to hNET and GAT-1 is saturated, the transporter remains partially active. This could be explained by a noncompetitive interaction of 2 with hNET and GAT-1, as both substrates and this lignan most likely bind to the transporter simultaneously. Compound 2 did not modulate transport of GABA through GAT-3, exhibiting an IC50 value greater than 500 μM. Although not as potent at inhibiting GAT-1 as other GAT-1 inhibitors such as tiagabine (0.07 μM), NNC-711 (0.04 μM), and CI 966 (0.26 μM),21 2 displayed additional activities toward the monoamine transporters, NET and DAT, which could be of a therapeutic interest for targeting multiple mechanisms in the therapy for ADHD associated with anxiety.32,33 The selectivity of 2 for these transporters was confirmed from the lack of modulation by this compound on the assays performed on glutamate transporters and glycine transporters. Dose−response curves of 2 at concentrations ranging from 0.5 to 500 μM did not inhibit glutamic acid transport by EAAT1−3 or glycine transport by GlyT1 (Table 1). The (−)-Hinokinin (2) Analogues Cubebin (1) and Methylcubebin (3) Display Diverse Properties on Monoamine Transporters. Owing to their similar structures, it was hypothesized that compounds 1 and 3 also modulate some of the monoamine transporters investigated. Dose−response curves for 3 (0.2−200 μM) indicated this compound to have a lower affinity for DAT compared to 2, with an IC50 value of 78.6 ± 2.4 μM (Figure 2A), suggesting the presence of a methoxy group and that the resultant increase in size is detrimental to the binding of this compound to hDAT. Even at the highest tested concentration, no effect of 3 was observed on NET activity, suggesting an even more detrimental effect of the methoxy group on affinity toward this carrier. hSERT was inhibited by 3 with similarly low affinity to 2 with a halfmaximal inhibitory concentration of 90.3 ± 30 μM (Figure 2A). Some intriguing effects of 1 were observed when compared with 2. At concentrations of 0.2−200 μM, 1 displayed a dose− response inhibition of dopamine transport with an IC50 value of 12 ± 0.04 μM (Figure 2B). This is very similar to the value observed for 2 with respect to hDAT. However, compound 1 showed low affinities for hNET and hSERT with IC50 values greater than 200 μM. This suggests that for the interaction with these two carriers, hydrogen bonding and increased hydro-

other diseases including orthostatic intolerance and depression.20 GABA is the main inhibitory neurotransmitter that is responsible for regulating neuronal excitability and information processing.21 Four GABA transporters (GATs) have been identified, with GAT-1 localized in neurons and astrocytes in the cerebral cortex.22 Current therapies that modulate GABA transporters are known to be effective in the treatment of epilepsy and anxiety. Tiagabine is a selective reuptake GAT-1 inhibitor that was approved for partial-onset seizures in adults; furthermore, this medication has been reported to be effective in the treatment of generalized anxiety disorder.23 Further characterization of compounds affecting the dopamine, norepinephrine, and GABA transporters is important because these sites have become established targets of pharmacotherapy for neurological conditions.24,25 These studies may lead to the development of new drugs for patients suffering with neurological and psychiatric disorders. The lignan (−)-cubebin (1), purified from the seeds of Piper cubeba L.f. (Piperaceae), has been found to display dosedependent effects on in vitro cultures of schistosomes beginning with the loss of parasite motility, followed by the separation of the worm pair, and finally a lethal effect.26 Considering a previous demonstration of the presence of monoamine transporters in the parasites (SmSERT and SmDAT)27,28 and also that monoamines play a vital role for parasite behavior inhibited by the cubebin derivatives, the effect of these compounds was studied on newly isolated schistosomal monoamine transporters. Transport assays were performed to investigate the effect of several different cubebin analogues on the parasite and the human biogenic amine transporters. In the present study, the modulatory effects were investigated for the lignans (−)-cubebin (1), (−)-hinokinin (2), and (−)-Omethylcubebin (3). Interestingly, these lignans have been described as having bacteriostatic activities against oral pathogens29 as well as fungicidal activity against Candida albicans.30 Additionally, (−)-cubebin derivatives have been shown to have analgesic and anti-inflammatory activities in male Swiss albino mice and male Wistar rats, respectively.31 Herein are described the specificity, selectivity, and mechanism of action of compound 2 on several different human neurotransmitter transporters, to examine its potential use in drug therapy. The inhibitory activity toward human biogenic amine transporters [hNET and hDAT] and GABA transporters (GATs) was determined for this compound. The mechanism of action by which this lignan derivative affects these transporters is described, and its potential future for therapeutic applications is conjectured.

■

RESULTS AND DISCUSSION (−)-Hinokinin (2) Inhibits Dopamine, Noradrenaline, and GABA Transport, but Not Glutamate and Glycine 1890

dx.doi.org/10.1021/np400452n | J. Nat. Prod. 2013, 76, 1889−1895

Journal of Natural Products

Article

Figure 1. (−)-Hinokinin (2) inhibits hDAT, hNET, and GAT-1 with low affinity for hSERT. Dose−response assays for the effects of 2 on monoamine and GABA transporters were performed in MDCK cells stably transfected with hDAT or hNET or in COS-7 cells transiently transfected with hNET or GAT-1. Dose−response curves shown are one representative curve of three independent assays for each transporter. (A) Dopamine transport assays generated an IC50 value of 8.35 ± 0.2 μM for the effect of 2. (B) Serotonin transport assays generated an IC50 value of 175 ± 3.7 μM for the effect of 2. (C) Noradrenaline transport assays generated an IC50 value of 8.76 ± 0.4 μM for the effect of 2. (D) GAT-1 transport assays generated an IC50 value of 20.6 ± 0.4 μM for the effect of 2.

philicity that results from the hydroxy group in 1 are detrimental for interactions with hNET and hSERT. Therefore, 2 is the most potent lignan of those tested and has a specific action for hDAT and hNET (with low specificity for hSERT), but also targets GAT-1. On the other hand, lignan 3 displayed significantly lower affinity for these transporters. Analogue 1 is also an hDAT inhibitor but did not show the propensity of 2 to interact with NET. Structure−activity relationship studies could be pursued to yield useful information regarding the further effects of functional groups within this lignan structural class, leading to the generation of analogues with higher affinity for these three transporters. (−)-Hinokinin (2) Noncompetitively Inhibits Dopamine and Noradrenaline Transport and Allosterically Modulates GABA Transport. Figure 3A shows the effect of 2 on kinetic parameters of dopamine transport. Compound 2 decreased the Vmax of dopamine transport when compared to vehicle by 63% (Vmax of 247.3 ± 14 pmol/mg/min in the absence of 2, compared to 80.2 ± 8.4 pmol/mg/min in the presence of 20 μM of this lignan, ***p < 0.001). No changes in the affinity for dopamine were observed: the KM was 10.8 ± 4.4 μM in the presence of 2, compared to 4.40 ± 1.07 μM with vehicle, indicating that (−)-hinokinin acts as a noncompetitive inhibitor of dopamine transport. The transport assays for the effects of 2 on the kinetics of norepinephrine uptake velocity

Table 1. Specificity of (−)-Hinokinin (2) on the Neurotransmitter Transporters Assayed transporter Monoamine transporters hDATb hNETb hSERTc GABA transporters GAT-1b GAT-3d Glutamate transporters EAAT1d EAAT2d EAAT3d Glycine transporters GlyT1d

IC50a (μM) 8.35 ± 0.2 8.76 ± 0.4 175 ± 3.7 20.6 ± 0.4 >500 >500 >100 >500 >500

a

IC50 values were calculated from at least three independent experiments performed in triplicate. bCompound 2 inhibited neurotransmitter transport by hDAT, hNET, and GAT-1. cCompound 2 inhibited neurotransmitter transport with low affinity for hSERT. d Compound 2 did not modulate glutamate transport mediated by EAAT1−3, glycine transport mediated by GlyT1, or GABA transport mediated by GAT-3.

1891

dx.doi.org/10.1021/np400452n | J. Nat. Prod. 2013, 76, 1889−1895

Journal of Natural Products

Article

Figure 2. Analogues of 2 modulate monoamine transporters differentially. Dose−response curves for the effects of (−)-cubebin (1) (A) and (−)-Omethylcubebin (3) (B) on the activity of biogenic monoamine transporters. Assays were performed in MDCK cells stably transfected with hDAT or hSERT or transiently transfected with hNET in COS-7 cells. Cells were incubated with various concentrations (0.2−200 μM) of 1 or 3 and 50 nM of radiolabeled substrates. Concentrations of compounds are plotted on a logarithmic scale. (A) Dopamine transport generated an IC50 value of 12 ± 0.4 μM for the effects of 1. Serotonin transport assays generated an IC50 value greater than 200 μM for hSERT, indicating low affinity. A dose− response curve showed that 1 does not affect norepinephrine transport velocity or substrate affinity. (B) The dose−response curve of 3 indicated low affinity for DAT with an IC50 value of 78.6 ± 2.4 μM. Serotonin transport assays generated an IC50 value of 93.8 μM for the effects of 3. Norepinephrine transport assays indicated no effect on NET. An uptake velocity of 100% corresponds to an absolute uptake rate of 106 ± 1.6 pmol/ mg/min for hDAT, 116 ± 5.3 pmol/mg/min for hNET, and 149 ± 4.2 pmol/mg/min for hSERT.

Figure 3. (−)-Hinokinin (2) inhibits hDAT and hNET noncompetitively and affects allosterically GAT-1. Kinetic analyses of dopamine, norepinephrine, and GABA transport in the presence of 2 were performed in hDAT MDCK stable transfected cells or COS-7 cells transiently transfected with hNET or GAT-1, which were preincubated in the absence (red) or presence (blue) of 20 μM 2. Data were from three independent uptake experiments using eight substrate concentrations. Statistical analysis on Vmax and KM in the presence or absence of 2 was performed using Student’s t-test for paired data using GraphPad Prism 5.03 (San Diego, CA, USA) (***p < 0.001; **p < 0.05).

generated 0.46 ± 0.13 μM for the KM and 112.7 ± 7.5 pmol/ mg/min for the Vmax under control conditions and 0.24 ± 0.08

μM for KM and 77.2 ± 5.4 pmol/mg/min for Vmax in the presence of 2. This indicated that (−)-hinokinin, similar to its 1892

dx.doi.org/10.1021/np400452n | J. Nat. Prod. 2013, 76, 1889−1895

Journal of Natural Products

Article

effects on hDAT, acts as a noncompetitive inhibitor of norepinephrine transport with a decrease of about 27% in Vmax (**p < 0.05) and no changes in substrate affinity. Hence, 2 reduces the efficiency of transport of dopamine and norepinephrine by interacting with the transporter at a different site from the binding site for dopamine or norepinephrine. Particularly in the case of hNET, this correlates well with the observation of a plateau in the dose−response assays in Figure 1. Several noncompetitive inhibitors have been characterized for the monoamine transporters. For example, the indole alkaloid and psychoactive substance ibogaine is a noncompetitive inhibitor of SERT.34 A peptide isolated from the venom of the predatory marine snail Conus marmoreus, χ-MrIA, is also a noncompetitive inhibitor of hNET and is currently being developed as a pain treatment.35 Neither of these two compounds shows chemical similarities to (−)-hinokinin, and unlike competitive inhibitors, the structural mechanism of action of these drugs is not well established. Figure 3C shows the effects of 2 on the kinetic parameters of GABA uptake velocity, generating a KM of 16.9 ± 3.8 μM and 1.4 ± 0.1 nmol/ mg/min for the Vmax under control conditions and a KM of 4.45 ± 0.94 μM and 0.7 ± 0.03 nmol/mg/min for Vmax in the presence of 2. This indicates that this lignan acts through a more complex mechanism that could involve allosteric modulation of the GAT-1 transporter, inhibiting both Vmax by approximately 50% (***p < 0.001) and decreasing KM 3- to 4fold (**p < 0.05). (−)-Hinokinin (2) Does Not Alter the Efflux of Dopamine, Noradrenaline, or GABA. Assays that examine reversed transport efflux are well established for testing if a compound is an actual substrate of the transporters. Efflux assays for dopamine, norepinephrine, and GAT-1 transporters were performed. The integrity of the assay was confirmed by the efflux elicited by non-radiolabeled substrate (Figure 4A−C, red bars). There is a good correlation between a compound’s IC50 value for inhibiting uptake and its EC50 value for elicting efflux, if it is a substrate. As the IC50 values observed for 2 against hDAT and hNET were close to 10 μM, it should elicit efflux at this concentration or any higher concentrations if it is a substrate. Compound 2 did not elicit dopamine efflux when compared to basal levels (vehicle) in MDCK GFP-hDAT cells at concentrations ranging from 0.5 to 500 μM (Figure 4A). (−)-Hinokinin lacked any effect on noradrenaline efflux (in COS-7 cells transfected with hNET) at the same concentrations (Figure 4B). Similarly, 2 did not alter GABA efflux in COS-7 cells transfected with GAT-1 at concentrations varying from 0.5 to 500 μM (Figure 4C), which included the concentration that inhibited GAT-1 uptake. The efflux assays conducted validate that 2 is an inhibitor of hDAT- and hNETmediated transport, and it does not act as a substrate for DAT or NET. Therefore, interestingly, 2 targets DAT and NET through a different mechanism of action from current drugs prescribed for ADHD. A different mechanism of action suggests that this compound might lead to alternative therapy for ADHD patients not responding optimally to their current medications. Interestingly, current drug therapies for ADHD are selective for the dopamine and norepinephrine transporters, but not the serotonin transporter.5 Unfortunately, treatments for ADHD have been reported to increase anxiety; therefore, (−)-hinokinin may be a potential pharmacotherapy lead compound to mitigate these symptoms through the inhibition of GAT-1.

Figure 4. (−)-Hinokinin (2) does not affect the release of dopamine, norepinephrine, or GABA. Efflux assays of dopamine in the presence of 2 were performed in MDCK-hDAT cells and for norepinephrine and GABA in hNET and GAT-1 transiently transfected COS-7 cells, respectively. Non-radiolabeled substrates elicited neurotransmitter release, indicating cell integrity, while 2 did not elicit release at the tested concentrations. Bar graphs shown are representative of three independent efflux assays. Statistical analyses were performed using one-way ANOVA and compared for statistical significance using Dunnett’s post hoc test (***p < 0.001). Data were analyzed on GraphPad Prism 5.03 for Windows (GraphPad, San Diego, CA, USA).

(−)-Hinokinin (2) is nontoxic for mammals, when examined specifically using the Wistar rat peripheral blood micronucleus test, and this lignan has no previously observed genotoxic effects.36 Therefore, this compound merits investigation in phenotypic assays in animal models of ADHD such as the neonatal 6-hydroxydopamine (6-OHDA)-lesioned rat and the spontaneously hypertensive rat model.37 1893

dx.doi.org/10.1021/np400452n | J. Nat. Prod. 2013, 76, 1889−1895

Journal of Natural Products

■

Article

MDCK cells plated in 24-well plates expressing the appropriate transporter (hDAT, hNET, GlyT1, or naı̈ve) were washed with PBSCM. Cells were then incubated for 10 min at room temperature with various concentrations of lignans 1−3 (as above for COS-7 cells) or vehicle. Uptake reactions were initiated by the addition of 50 nM radiolabeled substrate (dopamine, serotonin, norepinephrine, or glycine). For dopamine transport assays, a catechol-O-methyl transferase, RO 41-0960, was added to the buffer to prevent substrate degradation. Uptake was carried out for 10 min at room temperature. Reactions were terminated and cells were lysed as above. Radioactivity was quantified and dose−response curves were generated as above. For the assays in this study, the nonspecific transport (background) was obtained from COS-7 cells transfected with an empty vector (pcDNA3) or naı̈ve MDCK cells. Sister cultures were plated to obtain a background each time an assay was performed, and these values were subtracted from the total. The signal-to-noise ratio observed across the assays was between 1% and 3%. Previous characterization has demonstrated that the uptake rate measured under these conditions was linear over time and amount of protein within the range used (not shown). Calculations of IC50. Data generated from dose−response assays on the effects of 1−3 on monoamine transport (mediated by hNET, hDAT, and hSERT), glutamate transport (mediated by EAAT1−3), GABA transport (mediated by GAT-1 and GAT-3), and glycine transport (mediated by GlyT1) were fitted to a dose−response curve by nonlinear regression analysis using GraphPad Prism version 5.03 for Windows. For the effects of 1 and 3, data were normalized to the percentage of control (vehicle) for better visualization and comparison among compounds. IC50 values are given as means ± SEM of three independent assays for the effects of each lignan on each of the transporters. Saturation Analyses of Dopamine, Noradrenaline, and GABA Uptake Performed in the Presence of (−)-Hinokinin (2). Dose−response assays revealed that the lignans did not affect or had very low affinity for some of the transporters (Table 1). Only the transporters that were inhibited by 2 with IC50 values < 100 μM were selected to be further examined for the effects on the kinetic parameters: hDAT, hNET, and GAT-1. To determine the effects of 2 on the kinetic parameters of the dopamine transporter, MDCK cells expressing hDAT were placed in 24-well plates, as before. Cells were washed with PBS-CM and preincubated with 20 μM 2 for 10 min at room temperature. Uptake reactions were initiated by the addition of varying concentrations (45 nM to 100 μM), in triplicate, of a mixture of non-radiolabeled and radiolabeled dopamine (99.9%:0.1%). Uptake reactions were terminated and radioactivity was counted as before. Kinetic parameters of 2 on hNET and GAT-1 were analyzed on transiently transfected COS-7 cells. Cells were preincubated with 2 as indicated above. For hNET COS-7 cells, RO 41-0960 and ascorbic acid were added to the buffer to prevent degradation of norepinephrine. Uptake reactions were initiated by the addition of varying concentrations of non-radiolabeled and radiolabeled norepinephrine (99%:1%) or GABA (99.95%:0.05%), in triplicate for all assays, and the assays were terminated as before. Calculations of Effects of (−)-Hinokinin (2) on the Kinetic Parameters of Transport. Nonspecific uptake (from naı̈ve MDCK cells or pcDNA3 COS-7 cells) was subtracted, and the data were analyzed assuming Michaelis−Menten kinetics, using GraphPad Prism version 5.03 for Windows. Values of the maximum efficiency of transport by the transporters (Vmax) and the affinity for the substrate (KM) were determined from at least three independent assays. Statistical analysis on the values of Vmax and KM, obtained in each experiment in the presence or absence of 2, was performed using Student’s t-test for paired data. Values were considered to be significantly different when p < 0.05. Efflux Assays. Efflux assays in the presence of 2 were performed for the transporters that showed inhibition of uptake with an IC50 value of