Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA.
J Pharmacol Exp Ther. 2011 Mar;336(3):724-33. doi: 10.1124/jpet.110.172882. Epub 2010 Sep 28.
Lobeline attenuates the behavioral effects of methamphetamine via inhibition of the vesicular monoamine transporter (VMAT2). To increase selectivity for VMAT2, chemically defunctionalized lobeline analogs, including lobelane, were designed to eliminate nicotinic acetylcholine receptor affinity. The current study evaluated the ability of lobelane analogs to inhibit [³H]dihydrotetrabenazine (DTBZ) binding to VMAT2 and [³H]dopamine (DA) uptake into isolated synaptic vesicles and determined the mechanism of inhibition. Introduction of aromatic substituents in lobelane maintained analog affinity for the [³H]DTBZ binding site on VMAT2 and inhibitory potency in the [³H]DA uptake assay assessing VMAT2 function. The most potent (K(i) = 13-16 nM) analogs in the series included para-methoxyphenyl nor-lobelane (GZ-252B), para-methoxyphenyl lobelane (GZ-252C), and 2,4-dichlorphenyl lobelane (GZ-260C). Affinity of the analogs for the [³H]DTBZ binding site did not correlate with inhibitory potency in the [³H]DA uptake assay. It is noteworthy that the N-benzylindole-, biphenyl-, and indole-bearing meso-analogs 2,6-bis[2-(1-benzyl-1H-indole-3-yl)ethyl]-1-methylpiperidine hemifumarate (AV-1-292C), 2,6-bis(2-(biphenyl-4-yl)ethyl)piperidine hydrochloride (GZ-272B), and 2,6-bis[2-(1H-indole-3-yl)ethyl]-1-methylpiperidine monofumarate (AV-1-294), respectively] inhibited VMAT2 function (K(i) = 73, 127, and 2130 nM, respectively), yet had little to no affinity for the [³H]DTBZ binding site. These results suggest that the analogs interact at an alternate site to DTBZ on VMAT2. Kinetic analyses of [³H]DA uptake revealed a competitive mechanism for 2,6-bis(2-(4-methoxyphenyl)ethyl)piperidine hydrochloride (GZ-252B), 2,6-bis(2-(4-methoxyphenyl)ethyl)-1-methylpiperidine hydrochloride (GZ-252C), 2,6-bis(2-(2,4-dichlorophenyl)ethyl)piperidine hydrochloride (GZ-260C), and GZ-272B. Similar to methamphetamine, these analogs released [³H]DA from the vesicles, but with higher potency. In contrast to methamphetamine, these analogs had higher potency (>100-fold) at VMAT2 than DAT, predicting low abuse liability. Thus, modification of the lobelane molecule affords potent, selective inhibitors of VMAT2 function and reveals two distinct pharmacological targets on VMAT2.
洛贝林通过抑制囊泡单胺转运体（VMAT2）来减轻安非他命的行为效应。为了提高对 VMAT2 的选择性，设计了化学去功能化的洛贝林类似物，包括洛贝烷，以消除烟碱型乙酰胆碱受体亲和力。本研究评估了洛贝烷类似物抑制 [³H]二氢四苯并嗪（DTBZ）与 VMAT2 结合以及 [³H]多巴胺（DA）摄取到分离的突触小泡的能力，并确定了抑制机制。在洛贝烷中引入芳基取代基，保持了类似物对 VMAT2 上 [³H]DTBZ 结合位点的亲和力以及在评估 VMAT2 功能的 [³H]DA 摄取测定中抑制效力。该系列中最有效的（K(i) = 13-16 nM）类似物包括对甲氧基苯基正洛贝烷（GZ-252B）、对甲氧基苯基洛贝烷（GZ-252C）和 2,4-二氯苯基洛贝烷（GZ-260C）。类似物对 [³H]DTBZ 结合位点的亲和力与 [³H]DA 摄取测定中的抑制效力不相关。值得注意的是，N-苯并吲哚、联苯和吲哚骨架的介体 2,6-双[2-（1-苄基-1H-吲哚-3-基）乙基]-1-甲基哌啶富马酸盐（AV-1-292C）、2,6-双（2-（联苯-4-基）乙基）哌啶盐酸盐（GZ-272B）和 2,6-双[2-（1H-吲哚-3-基）乙基]-1-甲基哌啶单富马酸盐（AV-1-294））分别]抑制 VMAT2 功能（K(i) = 73、127 和 2130 nM），但对 [³H]DTBZ 结合位点几乎没有亲和力。这些结果表明，类似物在 VMAT2 上与 DTBZ 相互作用于替代位点。[³H]DA 摄取的动力学分析表明，2,6-双（2-（4-甲氧基苯基）乙基）哌啶盐酸盐（GZ-252B）、2,6-双（2-（4-甲氧基苯基）乙基）-1-甲基哌啶盐酸盐（GZ-252C）、2,6-双（2-（2,4-二氯苯基）乙基）哌啶盐酸盐（GZ-260C）和 GZ-272B）具有竞争性机制。与安非他命类似，这些类似物从囊泡中释放 [³H]DA，但效力更高。与安非他命不同，这些类似物在 VMAT2 上的效力（>100 倍）高于 DAT，预测滥用潜力低。因此，洛贝林分子的修饰提供了 VMAT2 功能的有效且选择性抑制剂，并揭示了 VMAT2 上的两个不同的药理学靶点。
