Parkinson disease drug screening based on the interaction between D₂ dopamine receptor and beta-arrestin 2 detected by capillary zone electrophoresis

Abstract

Parkinson’s disease is the second most common neurodegenerative disease in the world. Beta-arrestin-2 has been reported to be an important protein involved in D₂ dopamine receptor desensitization, which is essential to Parkinson’s disease. Moreover, the potential value of pharmacological inactivation of G protein-coupled receptor kinase or arrestin in the treatment of patients with Parkinson’s disease has recently been shown. We studied the interaction between D₂ dopamine receptor and beta-arrestin-2 and the pharmacological regulation of chemical compounds on such interaction using capillary zone electrophoresis. The results from screening more than 40 compounds revealed three compounds that remarkably inhibit the beta-arrestin-2/D₂ dopamine receptor interaction among them. These compounds are promising therapies for Parkinson’s disease, and the method used in this study has great potential for application in large-scale drug screening and evaluation.

Graphical abstract

Keywords

drug screening / D₂ dopamine receptor / beta-arrestin-2 / capillary zone electrophoresis / protein-protein interaction / Parkinson’s disease

INTRODUCTION

G protein-coupled receptors (GPCRs) form a superfamily of heptahelical proteins that mediate various biological processes, ranging from neurotransmission and hormonal control of virtually all physiological responses to perception of taste, smell, light, and pain (Kobilka and Deupi, 2007; Rosenbaum et al., 2009). Upon stimulation, heptahelical receptors undergo conformational changes that allow binding of heterotrimeric G proteins, leading to the activation of different effectors and signaling pathways (Kobilka and Deupi, 2007). GPCRs also control their own responsiveness by activating mechanisms that result in their desensitization (Gainetdinov et al., 2004; Whalen et al., 2011). One of the first steps in this process involves the functional uncoupling of the G proteins from the receptors. This process is fairly rapid (from seconds to minutes) and depends on receptor phosphorylation by intracellular second-messenger-regulated kinases and GPCR kinases (GRKs) (Premont et al., 1995; Premont and Gainetdinov, 2007).

Second-messenger-regulated kinases can phosphorylate both active and inactive receptors, yielding heterologous desensitization. In the latter case, receptors that have not been activated may become desensitized by the activation of distinct receptors. However, GRKs specifically phosphorylate only agonist-occupied receptors, resulting in homologous desensitization (Reiter and Lefkowitz, 2006; Kelly et al., 2008).

GRK phosphorylation of receptors is not sufficient for desensitization and instead serves to create high-affinity sites to promote the binding of arrestin proteins, which in turn guarantees desensitization by preventing further coupling to G proteins (Reiter and Lefkowitz, 2006). Four distinct mammalian arrestin proteins are known, two of which (visual and cone arrestins) are restricted to the phototransduction pathway (Lefkowitz and Shenoy, 2005). The somatic isoforms β-arrestin-1 (arrestin-2) and β-arrestin-2 (arrestin-3) are ubiquitously expressed and assumed to regulate signaling as well as internalization of many heptahelical receptors (Reiter and Lefkowitz, 2006; DeWire et al., 2007; Ma and Pei, 2007; Whalen et al., 2011).

Dopamine receptors (DRs), which can be classified into D₁ and D₂ subtypes based on their pharmacological and biochemical characteristics, belong to the GPCR superfamily (Andersen et al., 1990; Neve et al., 2004). D₂DR has been implicated in the pathophysiology and treatment of movement disorders, schizophrenia, and drug addiction (Emilien et al., 1999; Goldman-Rakic et al., 2004; Mehler-Wex et al., 2006). Parkinson’s disease is the second most common neurodegenerative disorder; it is caused by the degeneration of dopaminergic neurons in the substantia nigra (Olanow et al., 2006; Savitt et al., 2006). The lack of dopamine leads to disturbances in the basal ganglia circuitry and, consequently, to motor deficits. Dopamine replacement therapy with L-dopamine, the precursor of dopamine, is an effective way to correct motor abnormalities, but long-term therapy often results in reduced response to drug and motor complications, such as dyskinesia (Fahn et al., 2004; Dodd et al., 2005).

The desensitization of D₂DR is mediated by β-arrestin-2, which binds to the phosphorylated D₂DR to terminate the signal cascade (Oakley et al., 2000; Macey et al., 2004; Whalen et al., 2011). Suppression or inhibition of β-arrestin-2 could become an effective strategy to enhance the antinociceptive properties of analgesic morphine and reduce its side effects. In contrast, inactivation of the arrestin proteins could result in relatively amplified efficacy of endogenous or exogenous dopamine stimulation and is potentially helpful in restoring movement among patients with Parkinson’s disease.

Capillary electrophoresis (CE) is an important and powerful method to study protein–protein interactions (Ding et al., 2005; Kostal et al., 2008). Capillary zone electrophoresis (CZE) is probably the most common mode of CE. CZE involves the electrophoretic separation of analytes under constant temperature and pH conditions in free solution within a capillary. Compared with other methods, CE can obtain more concrete results over a short period with small samples (in nanograms) (Holzgrabe et al., 2006; Kostal et al., 2008).

As β-arrestin-2 is one of the key elements in the desensitization of D₂DR, inhibition of its activity may enhance the signaling from the upstream signal pathway (DeWire et al., 2007). The dopamine theory of Parkinson’s disease indicates that dopamine deficiency in the basal ganglia of the brain, which results in decreased signals from D₂DR, is the main cause of the disease (Obeso et al., 2008; Calabresi et al., 2009). Recent studies on the mechanism of receptor desensitization have proposed the use of β-arrestin-2 as a target for drug screening (Dromey and Pfleger, 2008; Levoye and Jockers, 2008). In this study, we used CZE to detect the interaction between D₂DR and β-arrestin-2. Large-scale drug screening indicated that three chemical compounds remarkably inhibited the said interaction, which renders them as potential drugs for the treatment of Parkinson’s disease.

RESULTS AND DISCUSSION

Detection of the D₂DR/β-arrestin-2 complex

The desensitization of GPCR depends on the D₂DR/β-arrestin-2 interaction (Macey et al., 2004; Skinbjerg et al., 2009). We used β-arrestin-2 as a target protein for drug screening against Parkinson’s disease. CE with laser-induced fluorescence (CE-LIF) has been proposed to detect and monitor protein–peptide and protein–protein interactions efficiently (García-Campaña et al., 2007; Yang et al., 2007). In this study, we detected a stable complex of FITC-arrestin/D₂DR using CE-LIF. The influence of different drugs on the complex could verify potential drugs for further screening. This method could be developed as an effective and simple instrument for large-scale drug screening and evaluation. Figure 1 shows the electropherograms of the FITC-arrestin/D₂DR complex detected by CE-LIF. CZE analysis of the single sample containing 2.71 μmol/L FITC-labeled β-arrestin-2 showed two peaks at 8.5 and 9.5 min, with the first peak representing FITC-arrestin and the second representing free FITC (Fig. 1A). However, CZE analysis of a mixture of 2.71 μmol/L FITC-labeled β-arrestin-2 with 1.26 μmol/L D₂DR identified three populations that migrated at 5.5, 8.5, and 9.5 min (Fig. 1B); the peak at 5.5 min represented the FITC-arrestin/D₂DR complex. As only β-arrestin-2 was labeled with FITC, FITC-labeled β-arrestin-2 (the peak at 8.5 min), free FITC (the peak at 9.5 min), and the FITC-arrestin/D₂DR complex (the peak at 5.5 min) were detected in chromatograms. CZE analysis of the mixture of FITC and D₂DR (at the same concentration but without incubation) was immediately performed to confirm whether the new peak, which appeared before the FITC-labeled β-arrestin-2, corresponded to the FITC-arrestin/D₂DR complex but not the FITC-labeled D₂DR. The results showed that FITC cannot bind to D₂DR in such a short period. Future experiments on the negative control protein of either mutant β-arrestin-2 or mutant D₂DR should demonstrate the specificity and functional relevance of this complex formation.

Pharmacological regulation of chemical compounds detected by CZE

More than 40 compounds, including phytic acid, 3-amino ethylamine, and 1,3-dichloro-2-propanol, were added to the mixtures to test the pharmacological regulation of the drugs on the D₂DR/β-arrestin-2 interaction. The mixtures were then injected into uncoated fused capillaries and separated by the different charges and sizes. We conveniently obtained information about the interaction from the electropherograms. Some compounds have acute effects on the native structures of the target proteins, whereas others have more moderate regulatory activities on the interaction, which showed more drug-like properties because the physiological regulations are usually reversible.

3-amino ethylamine

Many chemical compounds, such as 3-amino ethylamine, acutely influence target proteins, but most irreversible actions to biomacromolecules could result in cell damage. Figure 2 shows the electropherograms of 33 μmol/L 3-amino ethylamine added to the mixture of 2.71 μmol/L FITC-arrestin and 1.26 μmol/L D₂DR. Although the FITC-arrestin/D₂DR complex was completely inhibited, the mobility of all peaks significantly increased, suggesting that either the characteristics of the capillary were modified or this chemical compound affected the structures of the target proteins. Therefore, the chemical compounds that influenced the FITC-arrestin/D₂DR complex similarly to 3-amino ethylamine have less drug-like properties.

Phytic acid

Phytic acid (myo-inositol hexaphosphoric acid) is an abundant plant constituent that comprises 1%–5% of edible legumes, cereals, oil seeds, pollens, and nuts. It has been reported to bind to photoreceptor arrestin and block its interaction with rhodopsin (Wilson and Copeland, 1997). Such an interaction predicts that phytic acid could alter GPCR desensitization. We investigated this inhibitory action using CZE. Phytic acid (33 μmol/L) was added to the mixture of 2.71 μmol/L FITC-labeled β-arrestin-2 and 1.26 μmol/L D₂DR (Fig. 3C). Compared with the data illustrated in panels A and B of Fig. 3, the FITC-arrestin/D₂DR complex was remarkably inhibited by phytic acid, suggesting that phytic acid has a detectable interaction with β-arrestin-2 and thereby blocks further binding of β-arrestin-2 to D₂DR. Phytic acid is hence a promising therapy for Parkinson’s disease.

Ethylenediamine and 1,3-dichloro-2-propanol

When 33 μmol/L ethylenediamine was added to the mixture of 2.71 μmol/L FITC-labeled β-arrestin-2 and 1.26 μmol/L D₂DR, the peak of FITC-arrestin/D₂DR complex in the electropherogram disappeared (Fig. 4C), indicating that ethylenediamine strongly inhibited the D₂DR/β-arrestin-2 interaction. Therefore, such chemical compounds as ethylenediamine that can inhibit the interaction have potential for use in the treatment of Parkinson’s disease. The screening results demonstrated that 1,3-dichloro-2-propanol had inhibition properties similar to those of ethylenediamine. When 33 μmol/L or 67 μmol/L 1,3-dichloro-2-propanol (or 100 μmol/L 1,3-dichloro-2-propanol; data not shown) was added to the mixture of FITC-labeled β-arrestin-2 and D₂DR, the peak of FITC-arrestin/D₂DR complex in the electropherograms remarkably decreased; this peak decreased with increasing concentrations of the compound (Fig. 5C and 5D). Dose-response analysis (33–100 μmol/L) on the claimed inhibitor 1,3-dichloro-2-propanol, which is important for small-molecule inhibitor studies, was conducted. Overall, the results indicated that 1,3-dichloro-2-propanol remarkably inhibited the D₂DR/β-arrestin-2 interaction and is a promising drug for the treatment of Parkinson’s disease.

This study screened potential drugs for Parkinson’s disease using CZE, which is based on the D₂DR/β-arrestin-2 interaction. Validation experiments using more traditional binding assays, such as immunoprecipitation and GST pulldown, should be conducted to demonstrate the effectiveness of the compounds in the cells. More data on the screened compounds at the cell and animal model levels are clearly needed.

CONCLUSIONS

Parkinson’s disease is a chronic progressive neurodegenerative movement disorder (Jankovic, 2008). The typical drug screening strategy for it is based on the dopamine theory. Although L-dopamine-like drugs have improved the health of patients with Parkinson’s disease, they are associated with many adverse effects (Calabresi et al., 2009; Dagher and Robbins, 2009). Specifically, research has shown that long-term treatment with L-dopamine may trigger symptoms of schizophrenia. Alternatively, pharmacological inactivation of GRK or arrestin could provide an effective approach to amplify the efficacy of endogenous or exogenous dopamine stimulation and will be helpful in restoring movement among patients with Parkinson’s disease. Drug-designing targets in D₂DR-related proteins, such as arrestin or GRK, remain unexplored and should thus be evaluated. In this study, we utilized CZE to screen potential drugs and initially found three valuable chemical compounds that have significant dose-dependent effects on the D₂DR/β-arrestin-2 interaction. These compounds have promising applications in the treatment of Parkinson’s disease with reduced side effects. Although these compounds need to be studied further, CZE proved to be an effective and convenient method of drug screening. It may be used for large-scale drug screening and evaluation.

MATERIALS AND METHODS

Reagents and materials

D₂DR was purchased from Sigma-Aldrich (St. Louis, MO, USA). Chemical compounds, such as 1,3-dichloro-2-propanol and ethylenediamine, and plasmid-encoded rat β-arrestin-2 were kindly provided by Prof. Xiang Zhou (College of Chemistry and Molecular Sciences, Wuhan University) and Prof. Robert J. Lefkowitz (Department of Medicine, Duke University Medical Center), respectively. β-arrestin-2 was expressed in Escherichia coli and purified according to published procedures (Xiao et al., 2004). All the other reagents used were of analytical grade.

Apparatus

CE experiments were performed in a Beckman P/ACE MDQ apparatus (Beckman-Coulter, Fullerton, CA, USA) with a LIF detector (excitation at 488 nm; emission at 520 nm) and a liquid cooling system. The uncoated fused silica capillaries (Yongnian Optic Fiber, China) measured 30 cm × 25 μm i.d. with a 20-cm distance from the detector.

Solutions and sample preparation

The running buffer in CE experiments was 10 mmol/L NaH₂PO₄-Na₂HPO₄ buffer (pH 8.0). All the solutions and deionized water were filtered through 0.22-μm cellulose acetate membrane filters and stored at 4°C. The concentrations of proteins were determined using Bio-Rad protein assays.

Preparation of FITC-labeled β-arrestin-2

FITC was dissolved in deionized water to a final concentration of 0.1 mmol/L. An aliquot (0.1 mL) of 0.1 mmol/L FITC solution was mixed with 0.9 mL of 0.1 mmol/L β-arrestin-2 (100 μL diluted in 800 μL of 20 mmol/L sodium bicarbonate buffer, pH 9.0). The solution was mixed thoroughly, left at 4°C and in the dark overnight, and then diluted to the desired concentrations with the running buffer prior to the CE experiments.

CE experiments

A new capillary was flushed with 1 mol/L NaOH for 60 min and then with deionized water for 60 min. Between measurements, the capillary was flushed with 1 mol/L NaOH for 2 min, with deionized water for 2 min, and with running buffer for 3 min. The capillary chamber temperature was controlled at 25°C by the liquid cooling system. The field strength of the capillary was 500 V/cm in the normal polarity mode. The samples were pressure-injected into the capillary at 0.5 psi for 5 s.

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