Comparison of the enantiomers of (±)-doxanthrine, a high efficacy full dopamine D1 receptor agonist, and a reversal of enantioselectivity at D1 versus alpha2C adrenergic receptors

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Abstract

Parkinson's disease is a neurodegenerative condition involving the death of dopaminergic neurons in the substantia nigra. Dopamine D1 receptor agonists are potential alternative treatments to current therapies that employ L-DOPA, a dopamine precursor. We evaluated the pharmacological profiles of the enantiomers of a novel dopamine D1 receptor full agonist, doxanthrine (DOX) at D1 and α2C adrenergic receptors. (+)-DOX displayed greater potency and intrinsic activity than (−)-DOX in porcine striatal tissue and in a heterologous D1 receptor expression system. Studies in MCF7 cells, which express an endogenous human dopamine D1-like receptor, revealed that (−)-DOX was a weak partial agonist/antagonist that reduced the functional activity of (+)-DOX and dopamine. (−)-DOX had 10-fold greater potency than (+)-DOX at α2C adrenergic receptors, with an EC50 value of 4 nM. These findings demonstrate a reversed stereoselectivity for the enantiomers of DOX at D1 and α2C receptors and have implications for the therapeutic utility of doxanthrine.

Introduction

Parkinson's disease (PD) is a neurodegenerative condition that involves the selective degeneration of dopaminergic neurons in the substantia nigra, resulting in a dopamine deficiency in the basal ganglia, a brain area that is essential for initiation and control of voluntary movement. The degeneration of the nigrostriatal pathway leads to the classic symptoms of PD, including tremors, rigidity, and slowness of movement (Dawson and Dawson, 2003). The most widely-used treatment for PD is levodopa (L-DOPA), a dopamine precursor that crosses the blood brain barrier and is enzymatically converted into dopamine (Hurley and Jenner, 2006). Although L-DOPA is currently the “gold standard” for PD treatment, it produces both acute and long-term adverse effects and loses its efficacy in late stage PD.

Additional PD therapies include dopamine agonists that directly stimulate dopamine receptors in the striatum. The dopamine receptors are classified into two families. The dopamine D1 receptor family, comprised of the D1 and D5 receptors, stimulates adenylate cyclases through coupling with the stimulatory G proteins Gαs and Gαolf (Huang et al., 2001). The dopamine D2-like receptor family (D2, D3, and D4 receptors) couples with inhibitory G proteins Gαi/o, leading to inhibition of adenylate cyclase or modulation of other effectors (Neve et al., 2004). Currently available direct agonists for PD target the D2-like receptors and include bromocriptine, ropinirole, and pramipexole (Hurley and Jenner, 2006, Schapira et al., 2006). Dopamine D2 agonists appear primarily effective in early stage PD and as adjuncts to L-DOPA. Similar to L-DOPA, D2-like receptor agonists have been shown to produce dyskinesias (Jenner, 2003, Olanow et al., 2004).

Alternative pharmacological treatments under development for treating PD have included selective dopamine D1 receptor agonists (for review see Zhang et al., 2008). Dihydrexidine (DHX) was the first selective, full dopamine D1 receptor agonist, and displays moderate 10-fold selectivity for dopamine D1 versus dopamine D2 receptors (Brewster et al., 1990, Mottola et al., 1992). Taylor et al. (1991) first demonstrated that DHX was remarkably effective at reducing MPTP-induced Parkinson-like symptoms in monkeys. Additionally, in a small human patient trial ABT431, another selective D1 agonist, was demonstrated to be as efficacious as L-DOPA at alleviating the symptoms of PD (Rascol et al., 2001). These studies confirmed, contrary to conventional wisdom, the importance of the dopamine D1 receptor as a potential target for PD.

We recently described the synthesis and preliminary characterization of doxanthrine (DOX), a bioisostere of DHX that has improved selectivity for dopamine D1 receptors (Cueva et al., 2006). Racemic DOX displayed approximately 100-fold greater selectivity for D1 over D2 receptors (Cueva et al., 2006). We found that (+)-DOX displayed 200-fold selectivity for D1 over D2, whereas (−)-DOX possessed only 20-fold selectivity for D1 versus D2 receptors. These studies revealed that (+)-DOX is a potent full agonist at the recombinant human D1 dopamine receptor. Here we have extended our studies to examine the functional activity of both the (+) and (−) enantiomers of DOX using porcine D1-like receptors as well as a novel endogenous human D1-like dopamine receptor model. To validate our biochemical studies, we used an in vivo mouse model to investigate the biological activity of both the (+) and (−) enantiomers of DOX to produce changes in locomotor activity.

In addition, the initial study by the NIMH-sponsored Psychoactive Drug Screening Program revealed that racemic DOX possesses significant binding affinity for the α2C adrenergic receptor (Cueva et al., 2006). In view of the fact that α2 adrenergic receptors are responsible for control of blood pressure, blood flow, and neurotransmitter release (Brede et al., 2004, Docherty, 1998), it seemed likely that racemic DOX might have additional undesirable pharmacological targets. Thus, we also examined the functional properties of (±)-, (+)-, and (−)-DOX at the α2C adrenergic receptor using a recombinant heterologous system.

In this work we demonstrate that (+)-DOX is a potent dopamine D1 receptor agonist in several systems. By contrast, (−)-DOX is a weak partial agonist at D1 receptors, but exhibits potent agonist activity at α2C receptors. Thus, enantioselectivity for activity at the α2C receptor is reversed compared to the D1-like (and D2-like) receptors (Cueva et al., 2006). This finding is remarkable in view of the fact that the D1, D2, and α2 receptors are all Family A G protein-coupled receptors, presumably with a common rhodopsin-like structure, and has theoretical relevance to understanding the binding motifs of agonist ligands in these receptors. Of more practical significance, however, is the demonstration that (+)-DOX has clear superiority over racemic DOX as a potential therapeutic agent, where the off-target α2C adrenergic receptor activation of (−)-DOX in the racemate would produce unwanted side effects. This interesting example is illustrative of a case where the less active enantiomer in a racemate is not simply inert, or “less active,” but possesses an active pharmacology that diverges from that of its antipode.

Section snippets

Chemicals and reagents

[3H] Cyclic AMP (30 Ci/mmol) was purchased from Perkin-Elmer (Boston, MA, USA). Dopamine, clonidine, SCH23390, SKF38393, and isobutylmethylxanthine were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Forskolin was purchased from Tocris Bioscience (San Diego, CA, USA). Enantiomers of DOX were synthesized as described previously (Cueva et al., 2006). pcDNA3.1/V5/his TOPO human D1 dopamine receptor was a gift from Dr. Bryan Roth and pcDNA3.1-α2C was provided by Missouri S&T

(+)-DOX is a potent agonist at the dopamine D1 receptor

We initially compared stimulation of cyclic AMP accumulation by the enantiomers of DOX using a heterologous expression system of HEK cells that stably express the human dopamine D1 receptor. Consistent with our previous studies (Cueva et al., 2006), both enantiomers stimulated cyclic AMP accumulation in this system. The (+) enantiomer of DOX displayed full intrinsic activity (111 ± 3%) relative to dopamine with an EC50 of ca. 50 nM (Fig. 1). The (−) enantiomer of DOX displayed markedly reduced

Discussion

DOX is a recently synthesized analogue of DHX, a full dopamine D1 agonist with anti-parkinsonian activity in MPTP-treated non human primates (Cueva et al., 2006, Taylor et al., 1991). In the present studies, we have examined the pharmacological properties of the enantiomers of DOX at dopamine D1 and α2C adrenergic receptors. Initial experiments compared the functional activity of (+)-DOX and (−)-DOX, as well as the endogenous agonist dopamine, at the D1 dopamine receptor using a heterologous D1

Role of the funding source

This work was supported by the Showalter Trust Fund Award to DJR and VJW, a TRASK award from Purdue University, NIMH grants MH060397 (VJW) and MH42705 (DEN), and the NIMH PDSP program. The Showalter Trust, Purdue University, and the NIMH had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication.

Contributors

Julie Przybyla performed the pharmacological characterization of the enantiomers of doxanthrine using the heterologous D1 and α2C expression systems and MCF7 cell line. She also wrote the first draft of the manuscript and participated in the revision process. Juan P. Cueva performed the chemical synthesis of the enantiomers and racemic mixture of doxanthrine. Benjamin R. Chemel performed the studies evaluating the DOX enantiomers using the native porcine striatal tissue and α2C receptor cell

Conflict of interest

The authors certify that they have no conflict of interest and certify that this work has not been published elsewhere.

Acknowledgements

This work was supported by the Showalter Trust Fund Award to DJR and VJW, a TRASK award from Purdue University, NIMH grants MH060397 (VJW) and MH42705 (DEN), and the NIMH PDSP program.

We acknowledge Mr. Jason Conley who assisted with the preparation and proof-reading of the manuscript.

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