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.

通过多巴胺转运体（DAT）抑制多巴胺再摄取已被认为是可卡因产生其精神运动刺激作用的主要机制。为了进一步了解可卡因药理作用背后的分子机制，以及可卡因滥用背后的机制，结构-功能研究的方向是在分子水平上表征DAT蛋白。3- α -（二苯甲氧基）tropane （benztropine， BZT）类似物的设计、合成和评价为DAT提供了有效的选择性探针。结构-活性关系（SAR）的发展与那些描述可卡因，尽管显著的结构相似性。此外，在可卡因滥用的动物模型中，对许多BZT类似物的行为评估表明，这两类基于tropane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表现出有效的运动刺激，不能完全替代可卡因的鉴别刺激，也不能明显地在恒河猴身上自我施用。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种作用不一致。这些观察结果表明，血清素和去甲肾上腺素转运体以及毒蕈碱受体的不同结合谱可能对这些化合物的药理作用产生重大影响。此外，通过改变母体化合物的结构从而改变其物理性质，将直接影响药代动力学（PK）和药效学（PD）。在体外和体内模型中评估这些化合物，以获得这些药物的PK和PD谱，与可卡因相比，与一系列n -取代BZT类似物相比，这些化合物很容易穿透血脑屏障，但与可卡因相比，它们具有较慢的起效和持续时间，这是一个适合作为药物治疗药物开发的谱。此外，还合成了两个系列(1)新型(+)-2-取代-4′，4”-diF-BZTs和(2)新型n -取代-4′，4”-diF-BZTs，其n -取代基被设计成比前几代化合物亲脂性更低。对这两个系列化合物的体外评价表明，许多化合物在DAT中具有良好的耐受性，并且对该靶标具有高亲和力和选择性。DAT上的SAR进一步支持了我们之前的发现，即与可卡因类基于tropane的DAT抑制剂相比，bzt不具有相同的结构要求，也没有相同的结构修饰来优化DAT结合。然而，(+)-2取代的bzt在运动活动和药物识别方面首次表现出类似可卡因的作用。目前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，我们还以放射性和/或不可逆配体的形式合成了许多重要的分子工具。我们的基于tropane的DAT抑制剂的叠氮（光活化）和异硫氰酸酯衍生物的放射性碘化类似物已经合成，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。我们还合成了第一个半胱氨酸选择性MTS和mfz2 -12的马来酰亚胺类似物，这些类似物正在与半胱氨酸导向的DAT突变研究相结合，以表征这种可卡因样不可逆配体的结合域。在sigma受体拮抗剂rimcazole的基础上，设计并合成了[3-顺-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）胺取代立卡唑的咔唑环系统提高了对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上具有高亲和力结合，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更像利莫唑，并且在小鼠中没有表现出类似可卡因的行为特征。然后制备了另一系列类似物，其亲脂性降低，先导化合物的结合亲和力提高到Ki = 8.5 nM。这些化合物也被评估为sigma配体，并使用CoMFA进行了3D-QSAR研究，并比较了DAT和sigma受体的需求。这一系列化合物似乎在DAT和sigma都有双重作用，这可能在开发治疗可卡因滥用的药物中被利用。目前，选定的化合物正在WT和DATKO小鼠中进行条件位置偏好评估，以进一步阐明其作用机制。除了经典的药物设计和合成之外，我们还继续在计算化学方面的努力，利用比较分子场分析（CoMFA）方法和相应的DAT结合亲和力，对rimcazole类似物和一系列2-取代3-芳基tropanes进行了三维定量构效关系（3D-QSAR）研究。这些研究导致了新的2-取代的tropane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。