meso-Transdiene analogs inhibit vesicular monoamine transporter-2 function and methamphetamine-evoked dopamine release

Abstract

Lobeline, a nicotinic receptor antagonist and neurotransmitter transporter inhibitor, is a candidate pharmacotherapy for methamphetamine abuse. meso-Transdiene (MTD), a lobeline analog, lacks nicotinic receptor affinity, retains affinity for vesicular monoamine transporter 2 (VMAT2), and, surprisingly, has enhanced affinity for dopamine (DA) and serotonin transporters [DA transporter (DAT) and serotonin transporter (SERT), respectively]. In the current study, MTD was evaluated for its ability to decrease methamphetamine self-administration in rats relative to food-maintained responding. MTD specifically decreased methamphetamine self-administration, extending our previous work. Classical structure-activity relationships revealed that more conformationally restricted MTD analogs enhanced VMAT2 selectivity and drug likeness, whereas affinity at the dihydrotetrabenazine binding and DA uptake sites on VMAT2 was not altered. Generally, MTD analogs exhibited 50- to 1000-fold lower affinity for DAT and were equipotent or had 10-fold higher affinity for SERT, compared with MTD. Representative analogs from the series potently and competitively inhibited [(3)H]DA uptake at VMAT2. (3Z,5Z)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-106), the 3Z,5Z-2,4-dichlorophenyl MTD analog, had improved selectivity for VMAT2 over DAT and importantly inhibited methamphetamine-evoked DA release from striatal slices. In contrast, (3Z,5E)-3,5-bis(2,4-dichlorobenzylidene)-1-methylpiperidine (UKMH-105), the 3Z,5E-geometrical isomer, inhibited DA uptake at VMAT2, but did not inhibit methamphetamine-evoked DA release. Taken together, these results suggest that these geometrical isomers interact at alternate sites on VMAT2, which are associated with distinct pharmacophores. Thus, structural modification of the MTD molecule resulted in analogs exhibiting improved drug likeness and improved selectivity for VMAT2, as well as the ability to decrease methamphetamine-evoked DA release, supporting the further evaluation of these analogs as treatments for methamphetamine abuse.

Fig. 1.

Chemical structures of lobeline, MTD, and MTD analogs incorporating the phenylethylene moiety of MTD into the piperidine ring system with the addition of various phenyl ring substituents. For clarity of presentation, compounds are grouped according to structural similarity. Top, lobeline, MTD, and MTD analogs with no phenyl ring additions. Middle, MTD analogs with dichloro, methoxy, or methyl additions. Bottom, MTD analogs with heteroaromatic phenyl ring substitutions.

Fig. 2.

Incorporating the phenylethylene moiety of MTD into the piperdine ring of the analogs affords a novel more rigid molecule. For all analogs in the series, the phenylethylene substituents in the MTD structure (left) were incorporated into the piperidine ring system to afford analogs (right) with a similar number of carbons between the piperidine nitrogen and the phenyl rings. This structural change reduces the molecular weight and the number of rotational carbon bonds (curved arrows) from four in MTD to two in the MTD analogs, affording a novel, more conformationally restricted structure.

Fig. 3.

MTD dose-dependently decreases methamphetamine self-administration, without altering food-maintained responding. Top, dose-related effect of acute MTD on methamphetamine (METH) self-administration. Bottom, effect of the high dose of MTD (17.0 mg/kg) on food-maintained responding. Data are expressed as mean ± S.E.M. for number of methamphetamine infusions (0.05 mg/kg/infusion) or number of pellets earned during 60-min sessions (n = 5–6). **, p < 0.01 compared with control.

Fig. 4.

Structural modifications to MTD afford analogs with decreased affinity for DAT. Analogs are grouped according to structural similarity of the aromatic rings. Top, lobeline, MTD, and MTD analogs with no aromatic ring substituents. Middle, MTD analogs with dichloro, methoxy, or methyl aromatic substituents. Bottom, MTD analogs containing heteroaromatic rings. MTD is repeated in all three panels for purpose of comparison. Nonspecific [³H]DA uptake was determined in the presence of 10 μM GBR 12909. Control (CON) represents specific [³H]DA uptake in the absence of analog (35.0 ± 1.55 pmol/mg/min). Legends provide analogs in order from highest to lowest affinity. n = 4 rats/analog.

Fig. 5.

MTD analogs inhibit [³H]5-HT uptake into rat hippocampal synaptosomes. Analogs are grouped according to structural similarity of the aromatic rings. Top, lobeline, MTD, and MTD analogs with no aromatic ring substituents. Middle, MTD analogs with dichloro, methoxy, or methyl aromatic substituents. Bottom, MTD analogs containing heteroaromatic rings. MTD is repeated in all three panels for purpose of comparison. Nonspecific [³H]5-HT uptake was determined in the presence of 10 μM fluoxetine. Control (CON) represents specific [³H]5-HT uptake in the absence of analog (1.67 ± 0.09 pmol/mg/min). Legends provide compounds in order from highest to lowest affinity. n = 4 rats/analog.

Fig. 6.

MTD analogs inhibit [³H]DTBZ binding to vesicle membranes from rat whole brain preparations. Analogs are grouped according to structural similarity of the aromatic rings. Top, lobeline, MTD, and MTD analogs with no aromatic ring substituents. Middle, MTD analogs with dichloro, methoxy, or methyl aromatic substituents. Bottom, MTD analogs containing heteroaromatic rings. MTD is repeated in all three panels for purpose of comparison. Nonspecific [³H]DTBZ binding was determined in the presence of 10 μM Ro-4-1284. Control (CON) represents specific [³H]DTBZ binding in the absence of analog (5.01 ± 0.10 pmol/mg protein). Analogs are arranged in order from greatest potency to least potency. n = 4 rats/analog.

Fig. 7.

MTD analogs inhibit [³H]DA uptake into rat striatal vesicles. Analogs are grouped according to structural similarity of the aromatic rings. Top, lobeline, MTD, and MTD analogs with no aromatic ring substituents. Middle, MTD analogs with dichloro, methoxy, or methyl aromatic substituents. Bottom, MTD analogs containing heteroaromatic rings. MTD is repeated in all three panels for purpose of comparison. Nonspecific [³H]DA uptake was determined in the presence of 10 μM Ro-4-1284. Control (CON) represents specific vesicular [³H]DA uptake in the absence of analog (29.3 ± 1.38 pmol/mg/min). Legends provide compounds in order from highest to lowest affinity. n = 4 rats/analog.

Fig. 8.

Inhibition of [³H]DTBZ binding does not predict inhibition of [³H]DA uptake at VMAT2. Data presented are K_i values from analog-induced inhibition of [³H]DTBZ binding and [³H]DA uptake at VMAT2 (Figs. 6 and 7, respectively). Pearson's correlation analysis of these data revealed a lack of correlation (Pearson's r = 0.42; p = 0.13) between the ability of analogs to inhibit [³H]DTBZ binding and [³H]DA uptake at VMAT2.

Fig. 9.

Lobeline, MTD, and MTD analogs competitively inhibit [³H]DA uptake into vesicles prepared from rat striatum. Concentrations of lobeline (0.25 μM), MTD (0.23 μM), UKMH-105 (0.11 μM), and UKMH-106 (0.16 μM) approximated the K_i values for inhibiting [³H]DA uptake into isolated synaptic vesicles obtained from the data shown in Fig. 7. K_m (top) and V_max (bottom) values are mean ± S.E.M. *, p < 0.05 different from control; **, p < 0.01 different from control. n = 4–7 rats/analog).

Fig. 10.

UKMH-105 does not inhibit methamphetamine-evoked endogenous DA release from striatal slices. Fractional DA release represents the amount of DA in each 5-min sample. Slices were superfused with UKMH-105 after 10-min collection of basal samples, as indicated by the arrow, and analog remained in the buffer until the end of the experiment. Methamphetamine (METH; 5 μM) was added to the buffer for 15 min as indicated by the horizontal bar. Fractional release data are expressed as mean ± S.E.M. pg/ml/mg of the slice weight. n = 5 rats.

Fig. 11.

In a concentration-dependent manner, UKMH-106 inhibits methamphetamine-evoked DA release in striatal slices. Top, fractional DA release represents the amount of DA in each 5-min sample. Slices were superfused with UKMH-106 after 10-min collection of basal samples, as indicated by the arrow, and analog remained in the buffer until the end of the experiment. Methamphetamine (METH; 5 μM) was added to the buffer for 15 min as indicated by the horizontal bar. Bottom, concentration-response curve was derived from peak response data for each concentration of UKMH-106. Fractional release and peak response data are expressed as mean ± S.E.M. pg/ml/mg of the slice weight. For fractional release, *, p < 0.05 different from methamphetamine alone. For peak response, *, p < 0.05 different from peak response of methamphetamine alone (CON). n = 8 rats.