Novel Probes For The Dopamine Transporter

通过多巴胺转运蛋白（DAT）抑制多巴胺再摄取已被表征为可卡因产生其精神刺激和增强作用的主要机制。为了进一步了解可卡因滥用的分子机制，DAT蛋白的结构-功能研究已被导向在分子水平上表征。3-α-（二苯基甲氧基）托烷（benztropine，BZT）类似物的设计，合成和评价提供了有效的和选择性的探针DAT。结构-活性关系（SAR）已经开发，与可卡因所描述的，尽管显着的结构相似性。此外，在可卡因滥用的动物模型中，对许多BZT类似物的行为评价表明，这两类托烷类多巴胺摄取抑制剂具有不同的药理学特征。一般而言，我们先前的研究表明，BZT类似物在小鼠中未表现出有效的运动刺激，不能完全替代可卡因辨别刺激，并且在恒河猴或松鼠猴中不能明显自我施用。这些化合物在体外作为多巴胺摄取抑制剂通常比可卡因更有效，尽管它们在体内的作用与这种作用不一致。通过改变母体化合物的结构，从而改变其物理性质，直接影响药代动力学（PK）和药效学（PD）。与可卡因相比，在体外和体内模型中评价这些化合物以获得这些药剂与一系列N-取代的BZT类似物的PK和PD曲线，已经证明这些化合物容易穿透血脑屏障，但与可卡因相比，它们具有较慢的起效和作用持续时间，这是一种适合开发为药物治疗剂的特征，并且可能与它们缺乏可卡因样行为特征直接相关。正在进一步研究这类化合物的相关结构、药理作用和药效学，并开发这些药物作为潜在的可卡因滥用治疗剂。在这方面，我们进一步表征了我们的新的DAT配体，JHW 007（N-正丁基-4？四个？diF-BZT），并发现该化合物具有独特的药理学特性，因为它缓慢地与多巴胺转运蛋白结合，不仅在小鼠、大鼠或猴子中没有表现出可卡因样作用，而且在小鼠中完全阻断可卡因诱导的运动活动和可卡因辨别刺激。这些研究首次表明多巴胺摄取抑制剂可以阻止可卡因产生其精神兴奋作用，并进一步支持DAT体内占用率可以决定这些化合物的行为作用的假设。使用微透析进一步评估这种和其他N-取代的BZTs使我们能够将细胞外多巴胺的增加速率和水平与体内这些化合物与DAT的结合联系起来。最近的研究使用定点诱变揭示了BZTs，可卡因和其他结构多样的多巴胺摄取抑制剂之间结合域的差异。有趣的是，与其他可卡因样DAT抑制剂相比，BZT在WT和D79E DAT突变体中显示出独特的结合特征，在分子水平上提供了与其独特行为特征相关的结合相互作用差异的证据。除了开发用于体内研究的药物外，我们还合成了许多重要的放射性和/或不可逆配体形式的分子工具。我们已经合成了基于托烷的DAT抑制剂的叠氮基（光活化）和异硫氰基衍生物的放射性碘化类似物，目前正在与蛋白质水解和MALDI MS结合使用，以阐明这些化合物与DAT和SERT共价结合的跨膜结构域。我们还合成了一种高亲和力的荧光DAT配体，目前正被用来表征DAT在活的神经元细胞和作为荧光探针可视化多巴胺神经元干细胞。

The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant and reinforcing actions. In order to understand further the molecular mechanisms underlying cocaine abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine, BZT) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the BZT analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the BZT analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rhesus or squirrel monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. By varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) is directly affected. Evaluating these compounds in both in vitro and in vivo models to obtain PK and PD profiles on these agents, in comparison to cocaine, with a series of N-substituted BZT analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they have a slower onset and duration of action, which is a suitable profile for development as pharmacotherapeutics and may be directly related to their lack of cocaine-like behavioral profiles. Further investigation into correlating structure, pharmacological action and pharmacodynamics of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In this regard, we have further characterized one of our novel DAT ligands, JHW 007 (N-n-butyl-4?4?-diF-BZT), and discovered that this compound has a unique pharmacological profile in that it slowly associates with the dopamine transporter and not only demonstrates no cocaine-like actions in mice, rats or monkeys, but fully blocks cocaine-induced locomotor activity and the cocaine discriminative stimulus in mice. These studies are the first to show that a dopamine uptake inhibitor can prevent cocaine from producing its psychostimulant actions and further support the hypothesis that the rate of in vivo occupancy of the DAT can dictate the behavioral actions of these compounds. Further evaluation of this and other N-substituted BZTs using microdialysis has allowed us to relate the rate and levels of increasing extracellular dopamine, in vivo, with binding of these compounds to the DAT. Recent studies using site-directed mutagenesis has revealed differences in binding domains between the BZTs, cocaine and other structurally diverse dopamine uptake inhibitors. Interestingly, the BZTs showed a unique binding profile in the WT and D79E DAT mutants as compared to other cocaine-like DAT inhibitors providing evidence, at the molecular level, of binding interaction differences that correlate with their distinctive behavioral profiles. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of radioactive and/or irreversible ligands. Radioiodinated analogues of both azido (photoactivated) and isothiocyanato-derivatives of our tropane based DAT inhibitors have been synthesized and are currently being utilized in combination with proteolysis and MALDI MS to elucidate transmembrane domains at which these compounds bind covalently to both DAT and SERT. We have also synthesized a high affinity fluorescent DAT ligand that is currently being used to characterize the DAT in living neuronal cells and as a fluoroprobe for visualization of dopamine neurons in stem cells.