Elsevier

Bioorganic & Medicinal Chemistry

Volume 19, Issue 23, 1 December 2011, Pages 7044-7048
Bioorganic & Medicinal Chemistry

Trace amine-associated receptor 1 is a stereoselective binding site for compounds in the amphetamine class

https://doi.org/10.1016/j.bmc.2011.10.007Get rights and content

Abstract

The demonstrated ability of amphetamine to functionally activate the rat trace amine associated receptor 1 (rTAAR1) and the subsequent reports of amphetamine activation of TAAR1 in rhesus monkey mouse, human, and human-rat chimeric TAAR1-expressing cell lines has led to speculation as to the role of this receptor in the central nervous system (CNS) responses associated with amphetamine and its analogs. The agonist potencies of ten pairs of enantiomeric amphetamines, including several with known CNS activity, at primate TAAR1 stably expressed in RD-HGA16 cells, robustly indicate the S-configuration to be associated with higher potency. Moreover, the rank order of potency to activate TAAR1 parallels the stimulant action reported by humans for the specific amphetamines. Taken together, these data suggest that TAAR1 is a stereoselective binding site for amphetamine and that activation of TAAR1 is involved in the modulation of the stimulant properties of amphetamine and its congeners. In addition, the observed parallel between hTAAR1 and rhTAAR1 responses supports the rhesus monkey as a highly translational model for developing novel TAAR1-directed compounds as therapeutics for amphetamine-related addictions.

Graphical abstract

TAAR1 stereoselectivity for (S)- versus (R)-configuration for compounds in the amphetamine class supports a role for TAAR1 in the psychostimulant properties of this class of compounds and is consistent with TAAR1 as a stereoselective binding site for amphetamine.

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Introduction

Compounds in the amphetamine class have been known for decades to be highly addictive substances1 producing one or more of at least three distinct effects: an (S)-amphetamine-like effect, a DOM-like effect, and a PMMA-like effect.2 The DOM-like (hallucinogenic) effect has been associated with activation of the 5-HT2 family of receptors.3, 4, 5 In fact, the binding potencies of hallucinogenic amphetamines such as DOM, DOB, DOEt at 5HT2A/C receptors have been shown to correlate with measures of hallucinogenic potencies in rodent and the (R)-enantiomer has been found to possess higher potency than the (S)-enantiomer,6 consistent with binding data at 5-HT2A/C receptors. On the other hand, (S)-amphetamine, associated with stimulant properties, is bound at NET (EC50 = 7.1 nM) and DAT (EC50 = 24.8 nM) in cloned human cells7 and with low affinity at SERT (EC50 = 1.77 μM)7 and at rat adrenergic alpha sites (Ki∼1 μM)8 but not at 5-HT receptors. Binding sites for (S)-[3H]amphetamine have been reported in rat brainstem, hypothalamus, and striatum.9 Further studies have characterized two sites for [3H]amphetamine: a low affinity sequestration-site lacking stereospecificity10 and a high affinity, stereoselective site of (S)-[3H]amphetamine incorporation in striatal synaptosomes.11 However, these studies did not implicate either of these two [3H]amphetamine sites with (S)-amphetamine-like stimulant effects. The demonstrated ability of amphetamine to functionally activate the rat trace amine associated receptor 1 (rTAAR1)12 and the subsequent reports of amphetamine activation of TAAR1 in rhesus monkey,13 rat,14 mouse,14, 15, 16 human,15, 17 and human-rat chimeric14 TAAR1-expressing cell lines has led to speculation as to the role of this receptor in the psychostimulant, hallucinogenic and addictive effects associated with amphetamine and its analogs18 as well as to the suggestion that TAAR1 may contribute a novel mode of action to these hallucinogenic drugs.19 A recent study of the activation of mouse, rat, and human-rat chimeric TAAR1s by amphetamine, methamphetamine, and p-hydroxyamphetamine14 concluded that this receptor could be a mediator of the effects of these drugs.

We had expressed wild type hTAAR1 in CHO cells stably expressing Gα1617, 20 and had developed a high throughput assay for functional TAAR1 agonists20 that we have been using to evaluate a series of amphetamines. Here, we have similarly expressed rhTAAR1 and have collected binding data on a spectrum of stereoisomers of amphetamines, including several with known CNS activity, that demonstrate that primate TAAR1 is a stereoselective binding site for compounds in the amphetamine class. The findings suggest an opportunity for the rhesus as a valid model for assessing whether specific TAAR1-active agents may have therapeutic efficacy in humans.

Section snippets

Human TAAR1

A cell line expressing hTAAR1 was developed as previously described.20 Briefly, hTAAR1 cDNA was cloned from Marathon Ready cDNA from human stomach using the Advantage cDNA PCR kit (Clonetech, Mountain View, CA). The receptor coding sequence was amplified in 2 parts using primer pairs based on GenBank accession No. AF380185. The 2-part strategy was used because we had difficulty obtaining the full-length cDNA with 1 set of primers. The resultant PCR products were sub-cloned separately into the

Results

The potencies of ten pairs of enantiomeric amphetamines (110) to activate hTAAR117, 20 and rhTAAR1 stably expressed in RD-HGA16 cells, determined as previously described,20 are shown in Table 1. While all the data suggest possible stereoselectivity for the S-configuration (see Fig. 1), the data for activation of hTAAR1 are not compelling for five (2, 3, 5, 8, 10) of the ten compound pairs. However, the potency data for activation of rhTAAR1 robustly indicate the S-configuration to be preferred

Discussion

While a considerable body of quantitative data relative to potencies to activate m- and rTAAR1 has been reported,22, 23, 24, 25 particularly for thyronamine and its analogs, no such data are reported for rhTAAR1, and only scant data are available for hTAAR1. For the few compounds where cross species comparisons have been possible, differences between human and rodent responses to structural variations have been observed. For example, addition of a p-hydroxyl functionality to the aromatic ring

Acknowledgments

This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant: RO1-DA016327] (A.H.L.); National Institutes of Health National Institute on Drug Abuse [Grant: DA025802] (G.M.M. K02 salary support); The National Institutes of Health, National Center for Research Resources, New England Primate Research Center [Grant: RR00168] (G.M.M. base grant of NEPRC from NCRR); and National Institutes of Health National Institute of Neurological Disorders and Stroke

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