Novel Probes For The Dopamine Transporter

通过多巴胺转运体(DAT)抑制多巴胺再摄取已被认为是可卡因产生精神运动兴奋剂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，结构-功能研究一直致力于在分子水平上表征DAT蛋白。3-α-(二苯甲氧基)托烷(BZT)类似物的设计、合成和评价为DAT提供了有效和选择性的探针。结构-活性关系(SAR)已经被开发出来，尽管结构上有很大的相似之处，但与可卡因的描述形成了对比。此外，在可卡因滥用的动物模型中，对许多BZT类似物的行为评估表明，这两类基于托烷的多巴胺摄取抑制剂具有不同的药理学特征。总的来说，我们之前的研究表明，BZT类似物在小鼠身上没有显示出有效的运动刺激，不能完全替代可卡因的区别性刺激，在恒河猴或松鼠身上也不能明显地自我给药。在体外，这些化合物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一作用不一致。通过改变母体化合物的结构，从而改变它们的物理性质，直接影响药代动力学(PK)和药效学(PD)。与可卡因相比，使用一系列N取代的BZT类似物在体外和体内模型中对这些化合物进行评估，以获得这些药物的PK和PD曲线，结果表明，这些化合物很容易穿透血脑屏障，但与可卡因相比，它们的起效和持续时间更慢，这是一个适合作为药物治疗药物开发的曲线，可能与它们缺乏类似可卡因的行为曲线直接相关。对这类化合物的相关结构、药理作用和药效学的进一步研究正在进行中，并将这些药物开发为潜在的可卡因滥用疗法。在这方面，我们进一步鉴定了我们的一种新型DAT配体JHW 007(N-正丁基-4？4？-dif-BZT)，并发现该化合物具有独特的药理特征，它与多巴胺转运体缓慢结合，不仅在小鼠、大鼠或猴子身上没有可卡因样的作用，而且完全阻断可卡因诱导的小鼠的运动活动和可卡因的区别性刺激。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刺激作用，并进一步支持了DAT在体内的占有率可以决定这些化合物的行为行为的假设。使用微透析法对这种和其他N-取代的BZT进行进一步的评估，使我们能够将体内细胞外多巴胺的增加速度和水平与这些化合物与DAT的结合联系起来。最近使用定点突变的研究揭示了BZTS、可卡因和其他结构不同的多巴胺摄取抑制剂之间的结合结构域的差异。有趣的是，与其他类似可卡因的DAT抑制剂相比，BZT在WT和D79E DAT突变体中显示出独特的结合谱，这在分子水平上提供了与其独特的行为谱相关的结合作用差异的证据。除了开发用于体内研究的试剂外，我们还合成了一些重要的放射性和/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(光激活)和异硫氰酸衍生物的放射性碘类似物，目前正在结合蛋白质分解和MALDI MS来阐明这些化合物与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，目前正被用于表征活的神经细胞中的DAT，并作为荧光探针显示干细胞中的多巴胺神经元。

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.