Novel Probes For The Dopamine Transporter

多巴胺转运蛋白的新型探针

• 批准号: 6830613
• 负责人: Amy Hauck Newman
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON DRUG ABUSE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6830613
• 关键词: Macaca mulatta; analog; benztropine; chordate locomotion; cocaine; dopamine transporter; drug abuse; drug screening /evaluation; hormone receptor; laboratory mouse; matrix assisted laser desorption ionization; muscarinic receptor; neuropharmacology; neuroprotectants; pharmacokinetics; protein structure function; proteolysis; receptor binding; reinforcer; serotonin receptor

The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant actions. In order to understand further the molecular mechanisms underlying the pharmacological actions of cocaine, as well as mechanisms that underlie its 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 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. These observations suggest that differing binding profiles at the serotonin and norepinephrine transporters as well as at muscarinic receptors might have significant impact on the pharmacological actions of these compounds. In addition, by varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) will be 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. In addition, the synthesis of two additional series (1) novel (+)-2-substituted-4',4"-diF BZTs and (2) novel N-substituted 4',4"-diF-BZTs, whose N-substituents were designed to be less lipophilic then previous generations of compounds were prepared. In vitro evaluation of both series of compounds demonstrated that many were well tolerated at the DAT and showed both high affinity and selectivity for this target. SAR at the DAT further supports our previous findings wherein the BZTs do not share structural requirements nor the same structural modifications to optimize DAT binding, as compared to the cocaine class of tropane-based DAT inhibitors. Nevertheless, for the first time, the (+)-2-substituted BZTs did demonstrate cocaine-like actions in both locomotor activity and drug discrimination. Further investigation into correlating structure and pharmacological action of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. 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 the first cysteine-selective MTS and maleimide analogs of MFZ 2-12, that are being used in combination with cysteine-directed mutagenesis studies of the DAT to characterize the binding domain of this cocaine-like irreversible ligand. In another series of compounds, based on the sigma receptor antagonist, rimcazole, [3-cis-3,5-dimethyl-1-piperazinyl)alkyl] bis-(4'-fluorophenyl)amines were designed and synthesized. We found that substitution of the carbazole ring system of rimcazole with bis-(4'-fluorophenyl)amine improves binding affinity and selectivity for the DAT. The most potent compound in this series to date showed a DAT binding affinity of (Ki=17.5 nM) which is comparable to GBR 12909. Despite high affinity binding at DAT, and structural similarity to GBR 12909, preliminary studies suggest that these compounds behave more like rimcazole than GBR 12909 and do not demonstrate a cocaine-like behavioral profile in mice. Another series of analogues were then prepared in which lipophilicity was decreased and binding affinity of the lead compound improved to Ki = 8.5 nM. These compounds have also been evaluated as sigma ligands and 3D-QSAR studies using CoMFA and comparing DAT and sigma receptor requirements have been undertaken. This series of compounds appear to have dual actions at both DAT and sigma, which may be exploited in the development of agents to treat cocaine abuse. Currently, selected compounds are being evaluated for conditioned place preference in WT and DATKO mice to further elucidate the mechanism of their actions. In addition to classical drug design and synthesis, we have continued our computational chemistry efforts with a three dimensional quantitative structure-activity relationship (3D-QSAR) study on both the rimcazole analogues as well as a series of 2-substituted 3-aryltropanes, using the comparative molecular field analysis (CoMFA) method with their corresponding DAT binding affinities. These studies have led to the design of novel 2-substituted tropane and rimcazole analogues with which we will continue to elucidate structure and function of the binding sites of the DAT.

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兴奋作用的主要机制。为了进一步了解可卡因药理作用的分子机制，以及其滥用的机制，结构-功能研究已被导向在分子水平上表征DAT蛋白。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类似物在小鼠中没有表现出有效的运动刺激，不能完全替代可卡因辨别刺激，并且在恒河猴中没有明显的自我给药。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。这些观察结果表明，在血清素和去甲肾上腺素转运蛋白以及在毒蕈碱受体的不同结合概况可能对这些化合物的药理作用有显着影响。此外，通过改变母体化合物的结构，从而改变其物理性质，药代动力学（PK）以及药效学（PD）将受到直接影响。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物与一系列N-取代的BZT类似物的PK和PD特征，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，这是开发为药物治疗剂的合适特征。此外，制备了另外两个系列的合成：（1）新的（+）-2-取代的-4 '，4”-diF BZT和（2）新的N-取代的4'，4”-diF-BZT，其N-取代基被设计成比前几代化合物更不亲脂。这两个系列的化合物的体外评价表明，许多在DAT下耐受良好，并显示出对该靶标的高亲和力和选择性。DAT的SAR进一步支持了我们先前的发现，其中与可卡因类托烷类DAT抑制剂相比，BZT不具有相同的结构要求，也不具有相同的结构修饰以优化DAT结合。尽管如此，第一次，（+）-2-取代的BZTs确实表现出可卡因样的行动，在运动活动和药物辨别。正在进一步研究这类化合物的相关结构和药理作用，并开发这些药物作为潜在的可卡因滥用治疗药物。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。已经合成了我们的基于托烷的DAT抑制剂的叠氮基（光活化的）和异硫氰酸根合衍生物的放射性碘化类似物，并且目前正与蛋白水解和MALDI MS组合使用，以阐明这些化合物共价结合DAT和SERT的跨膜结构域。我们还合成了MFZ 2-12的第一个半胱氨酸选择性MTS和马来酰亚胺类似物，它们与DAT的半胱氨酸定向诱变研究结合使用，以表征这种可卡因样不可逆配体的结合结构域。在另一系列化合物中，基于σ受体拮抗剂瑞姆卡唑，设计并合成了[3-顺式-3，5-二甲基-1-哌嗪基）烷基]双-（4 '-氟苯基）胺。我们发现，用双-（4 '-氟苯基）胺取代rimcazole的咔唑环系统提高了DAT的结合亲和力和选择性。迄今为止，该系列中最有效的化合物显示出与GBR 12909相当的DAT结合亲和力（Ki=17.5 nM）。尽管在DAT处具有高亲和力结合，并且与GBR 12909结构相似，但初步研究表明，这些化合物的行为比GBR 12909更像林卡唑，并且在小鼠中未表现出可卡因样行为特征。然后制备另一系列类似物，其中亲脂性降低，先导化合物的结合亲和力提高至Ki = 8.5 nM。这些化合物也被评估为σ配体和3D-QSAR研究，使用CoMFA和比较DAT和σ受体的要求已经进行。这一系列的化合物似乎在DAT和sigma两者上具有双重作用，其可用于开发治疗可卡因滥用的药剂。目前，正在WT和DATKO小鼠中评估选定化合物的条件性位置偏好，以进一步阐明其作用机制。除了经典的药物设计和合成，我们继续我们的计算化学的努力与三维定量构效关系（3D-QSAR）的研究都rimcazole类似物以及一系列的2-取代的3-芳基托烷，使用比较分子场分析（CoMFA）方法与其相应的DAT结合亲和力。这些研究导致了新的2-取代托烷和rimcazole类似物的设计，我们将继续阐明DAT结合位点的结构和功能。