JPET #139675 1 Studies of the Biogenic Amine Transporters. 12. Identification of Novel Partial

49 Pages·2008·0.87 MB·English

JPET Fast Forward. Published on April 25, 2008 as DOI: 10.1124/jpet.108.139675
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JPET #139675
Studies of the Biogenic Amine Transporters. 12. Identification of Novel Partial Inhibitors
of Amphetamine-Induced Dopamine Release
Joseph J. Pariser, John S. Partilla, Christina M. Dersch, Subramaniam Ananthan, and
Richard B. Rothman
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Clinical Psychopharmacology Section, IRP, NIDA, NIH, DHHS, Baltimore, MD (J.J.P., tjo
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J.S.P., C.M.D., R.B.R.) and Organic Chemistry Department, Southern Research ls
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Institute, Birmingham, AL 35255 (S.A.) t A
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Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on April 25, 2008 as DOI: 10.1124/jpet.108.139675
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Running Title: Allosteric modulators of DAT
Correspondence and Requests for Reprints to:
Richard B. Rothman, M.D., Ph.D.
NIH/NIDA
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Suite 4500, Triad Bldg. ed
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Email: [email protected] E
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Number of: on
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Text Pages 39 rua
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Tables 7 , 20
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Figures 10
References 22
Words in the Abstract, 210
Words in Introduction 479
Words in Discussion 951
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List of non-standard abbreviations:
SoRI-20040 N-(2,2-Diphenylethyl)-2-phenyl-4-
quinazolinamine
SoRI-20041 N-(3,3-Diphenylpropyl)-2-phenyl-4-
quinazolinamine
SoRI-2827 [4-Amino-6-[(diphenylmethyl)amino]-5-
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nitro-2-pyridinyl]carbamic acid ethyl ester n
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SoRI-9804 N-(Diphenylmethyl)-2-phenyl-4- fro
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quinazolinamine e
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SoRI-6238 [5-amino-3-(3,4-dichlorophenyl)-1,2- tjo
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dihydropyrido[3,4-b]pyrazin-7-yl]carbamic ls.o
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acid ethyl ester t A
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RTI-55 3β-(4'-iodophenyl)tropan-2β-carboxylic J
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acid methyl ester ls o
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TB-1-099 4-(2-[bis(4-fluorophenyl)methoxy]ethyl)-1- ru
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(2-trifluoromethyl-benzyl)-piperidine , 2
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GBR12909 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-
(3-phenylpropyl)piperazine
[125I]RTI-55 3β-(4'-125iodophenyl)tropan-2β-carboxylic
acid methyl ester
hSERT cloned human 5-hydroxytryptamine
transporter
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hNET cloned human norepinephrine transporter
hDAT cloned human dopamine transporter
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Abstract
Previous studies identified partial inhibitors and allosteric modulators of 5-HT ([5-amino-
3-(3,4-dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-yl]carbamic acid ethyl ester
[SoRI-6238], 4-(2-[bis(4-fluorophenyl)methoxy]ethyl)-1-(2-trifluoromethyl-benzyl)-
piperidine [TB-1-099]) and dopamine transporters N-(Diphenylmethyl)-2-phenyl-4-
quinazolinamine, [SoRI-9804]). We report here the identification of three novel allosteric
modulators of the dopamine transporter (N-(2,2-Diphenylethyl)-2-phenyl-4- D
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quinazolinamine [SoRI-20040], N-(3,3-Diphenylpropyl)-2-phenyl-4-quinazolinamine ad
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[SoRI-20041], [4-Amino-6-[(diphenylmethyl)amino]-5-nitro-2-pyridinyl]carbamic acid m
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ethyl ester, [SoRI-2827]). Membranes were prepared from HEK cells expressing the t.as
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cloned human dopamine (hDAT) transporter. [125I]RTI-55 binding and other assays urn
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followed published procedures. SoRI-20040, SoRI-20041 and SoRI-2827 partially rg
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inhibited [125I]RTI-55 binding with EC values ranging from ~1.4 µM to 3 µM and E SP
50 MAX E
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values decreasing as the [125I]RTI-55 concentrations increased. All three compounds u
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decreased the [125I]RTI-55 B and increased the apparent K in a manner well on
max d F
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described by a sigmoid dose-response curve. In dissociation rate experiments, SoRI- a
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20040 (10 µM) and SoRI-20041 (10 µM), but not SoRI-2827 (10 µM), slowed the 0
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dissociation of [ I]RTI-55 from hDAT by ~30%. Using rat brain synaptosomes, all
three agents partially inhibited [3H]dopamine uptake with EC values ranging from 1.8
50
µM to 3.1 µM and decreased the V value in a dose-dependent manner. SoRI-9804
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and SoRI-20040 partially inhibited amphetamine-induced DAT-mediated release of
[3H]MPP+ from rat caudate synaptosomes in a dose-dependent manner. Viewed
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collectively, we report several compounds that allosterically modulate hDAT binding and
function, and identify novel partial inhibitors of amphetamine-induced dopamine release.
.
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JPET Fast Forward. Published on April 25, 2008 as DOI: 10.1124/jpet.108.139675
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Introduction
The biogenic amine transporters for dopamine (DAT), norepinephrine (NET) and
serotonin (SERT), are important targets for a wide range of medications used to treat a
variety of psychiatric conditions such as anxiety, depression and obsessive compulsive
disorder (Gorman and Kent, 1999; Zohar and Westenberg, 2000). Drugs that interact
with transporters generally interact with these proteins in two distinct ways. Reuptake
inhibitors bind to transporter proteins but are not transported. These drugs elevate
extracellular concentrations of transmitter by blocking transporter-mediated uptake of
transmitters from the synapse. Substrate-type releasers bind to transporter proteins and
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are subsequently transported into the cytoplasm of nerve terminals, releasing n
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neurotransmitter via a process of carrier mediated exchange (Rudnick and Clark, 1993; e
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Rothman and Baumann, 2006). m
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There is growing interest in the possible therapeutic potential of allosteric t.a
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modulators (Christopoulos and Kenakin, 2002; Schwartz and Holst, 2007), including the tjo
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identification of allosteric modulators of the biogenic amine transporters (BATs) rna
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(Sanchez, 2006). Early evidence of allosteric interactions at the biogenic amine rg
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transporters included our finding that pre-treatment of guinea pig membranes with S
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paroxetine increased the dissociation rate of [3H]cocaine from SERT (Akunne et al., T J
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1992). Using rat SERT expressed in HEK cells, Sur et al. (Sur et al., 1998) presented rn
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evidence that imipramine allosterically modulated the ability of citalopram to inhibit n
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[3H]5-HT transport. Others reported apparent allosteric interactions between 5-HT and bru
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[3H]paroxetine binding to human platelet SERT (Andersson and Marcusson, 1989) and 1
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between β-estradiol and SERT (Chang and Chang, 1999). More recently, we reported 23
novel allosteric modulators of both DAT (N-(Diphenylmethyl)-2-phenyl-4-
quinazolinamine [SoRI-9804]) (Rothman et al., 2002) and SERT ([5-amino-3-(3,4-
dichlorophenyl)-1,2-dihydropyrido[3,4-b]pyrazin-7-yl]carbamic acid ethyl ester [SoRI-
6238], 4-(2-[bis(4-fluorophenyl)methoxy]ethyl)-1-(2-trifluoromethyl-benzyl)-piperidine
[TB-1-099]) (Nandi et al., 2004; Nightingale et al., 2005). Moreover, Chen et al. reported
evidence for allosteric modulation of [3H]-S-citalopram binding (Chen et al., 2005).
In 1999 we searched a library of compounds maintained by Southern Research
Institute for compounds that possessed a diphenylmethyl (benzhydryl) group. Using rat
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JPET Fast Forward. Published on April 25, 2008 as DOI: 10.1124/jpet.108.139675
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brain tissue assays, we screened these compounds for activity in binding assays for
DAT, SERT and NET (unpublished data). This effort identified several possible allosteric
modulators of the BATs. We examined in greater detail the interaction of selected
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agents with the BATs. SoRI-9804 (Fig. 1) partially inhibited [ I]RTI-55 binding to DAT
and partially inhibited [3H]DA uptake by rat brain synaptosomes. SoRI-6238, and a
subsequent compound that was not part of the SoRI library (TB-1-099), were shown to
allosterically modulate SERT (Nandi et al., 2004; Nightingale et al., 2005). In the
present study, we focused on three additional compounds identified as being potential
allosteric modulators (Fig. 1): SoRI-20040 (N-(2,2-Diphenylethyl)-2-phenyl-4-
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quinazolinamine), SoRI-20041 (N-(3,3-Diphenylpropyl)-2-phenyl-4-quinazolinamine) w
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and SoRI-2827 ([4-Amino-6-[(diphenylmethyl)amino]-5-nitro-2-pyridinyl]carbamic acid d
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ethyl ester). Initial screens indicated that all three agents were inactive at NET and fro
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SERT binding (IC50 values > 10 µM), but inhibited [125I]RTI-55 binding to the rat brain et.a
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DAT in a manner suggestive of allosteric interactions. We report here that these three etjo
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agents allosterically modulate the human DAT (hDAT) expressed in HEK cells and rna
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noncompetitively inhibit [3H]DA uptake by rat caudate synaptosomes. rg
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Methods
Animals. Male Sprague-Dawley rats (300-450 g), used for [3H]neurotransmitter uptake
assays, were obtained from Charles River Laboratories (Wilmington, MA). The animal
housing facilities were fully accredited by the American Association of the Accreditation
of Laboratory Animal Care (AAALAC), and all experiments were performed within the
guidelines delineated in the Institutional Care and Use Committee of the National
Institute on Drug Abuse (NIDA), Intramural Research Program (IRP).
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Tissue Preparation. HEK cells expressing hDAT were grown to confluency on plates, d fro
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using published methods (Nightingale et al., 2005). The medium was removed and the jp
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plates were stored at –800 C until the day of the assay. The plates were thawed, the etjo
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cells scraped off and rinsed with 55.2 mM sodium phosphate buffer, pH 7.4 (“BB”), and ls
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homogenized with a polytron at setting 6 for 10 sec. The homogenate was centrifuged t A
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twice at 30,000 x g for 10 min, with resuspensions in BB. The final pellets were J
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resuspended in a final volume of 225 ml/plate with BB. ls
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Transporter binding assays. For assays using HEK cells expressing hDAT, experiments 1
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were carried out in 12 x 75 mm polystyrene tubes that were pre-filled with 100 µL of
drug, 100 µL of radioligand, and 50 µL of a blocker or buffer (Rothman et al., 1998).
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[ I]RTI-55 was diluted in a protease inhibitor cocktail (BB with 25 µg/mL chymostatin,
25 µg/mL leupeptin, 1 mM EGTA, and 1 mM EDTA). The drugs and blockers were
made up in BB with 1 mg/mL BSA at pH 7.4. The experiment was initiated with the
addition of 750 µL of membranes in BB. Samples were incubated in a final volume of 1
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JPET Fast Forward. Published on April 25, 2008 as DOI: 10.1124/jpet.108.139675
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mL, at 0oC for 18-24 hrs (steady state). After incubation the samples were filtered with a
Brandel cell harvester over Whatman GF/B filters presoaked in wash buffer (ice-cold 10
mM Tris/HCl, 150 mM NaCl, pH 7.4) containing 2% poly(ethyleneimine). Typical total
and nonspecific cpms observed for the HEK cell transporter binding assays were 2000
and 75, respectively.
Kinetic experiments. Kinetic experiments were conducted with minor modifications of
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published methods (Nandi et al., 2004). For hDAT dissociation experiments, [125I]RTI- nlo
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55 (0.01 nM) was incubated for 2 hr at 25o C (steady state). At that point, 100 µL of RTI- fro
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55 (final concentration 1 µM) or buffer was added. Ten minutes later, test drug (10 µM)) e
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was added. This design insured that any effect of the test drug could not be due to tjo
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interactions with the DAT binding site, since this site would be completely occupied by ls.o
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RTI-55. Triplicate samples were filtered at various time points after the addition of test t A
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drug: 5,15,25,35, and 55 mins. For the purpose of data analysis (described below), the J
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100% of control point was “time 0 min” for conditions that did not receive test drug, and ls
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“time 10 min” for conditions that received test drug. b
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Neurotransmitter uptake assays. [3H]DA uptake inhibition assays were conducted as
described elsewhere (Rothman et al., 2001). Freshly removed caudate ([3H]DA uptake)
was homogenized in 10% ice-cold sucrose with 12 strokes of a hand-held Potter-
Elvehjem homogenizer followed by centrifugation at 1000 x g for 10 min. The
supernatants were saved on ice and used immediately. The assay buffer used was
Krebs-phosphate buffer containing 154.4 mM NaCl, 2.9 mM KCl, 1.1 mM CaCl , 0.83
2
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