Novel Probes For Monoamine Transporters

单胺转运蛋白的新型探针

• 批准号: 8336423
• 负责人: Amy Hauck Newman
• 金额: $ 58.23万
• 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/8336423
• 关键词: Affect; Affinity; Allosteric Site; Animal Model; Antidepressive Agents; Area; Attention; Attention deficit hyperactivity disorder; Attenuated; Behavior; Behavioral; Benztropine; Binding; Blood - brain barrier anatomy; Cell Line; Cells; Citalopram; Clinic; Cocaine; Cocaine Abuse; Computer Analysis; Data; Development; Disease; Dopamine; Dopamine Uptake Inhibitors; Drug Kinetics; Drug Receptors; Escitalopram; Evaluation; Food; Goals; Homologous Gene; Human; In Vitro; Investigation; Label; Lead; Life; Ligands; Macaca mulatta; Marketing; Microdialysis; Modafinil; Molecular; Molecular Conformation; Molecular Models; Mus; Narcolepsy; Neurons; Oxazoles; Parents; Pharmaceutical Preparations; Pharmacodynamics; Proteins; Rattus; Reporting; Research; Role; Saimiri; Self Administration; Self-Administered; Series; Site; Site-Directed Mutagenesis; Sleep Disorders; Stimulus; Structure; Structure-Activity Relationship; System; Testing; Therapeutic; Tropanes; Work; addiction; analog; base; computer studies; design; dopamine transporter; ecstasy; enantiomer; extracellular; improved; in vivo; in vivo Model; inhibitor/antagonist; interest; methamphetamine abuse; molecular modeling; monoamine; mutant; neuropsychiatry; noradrenaline transporter; novel; physical property; preference; psychostimulant; receptor; research study; reuptake; serotonin transporter; stimulant abuse; tool; trafficking; water solubility

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) 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 benztropine 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 benztropine 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 rats, 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. As such, we have described this class of compounds as atypical dopamine uptake inhibitors. By varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) are 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 benztropine analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they typically have a slower onset and longer 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 PD of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In this regard, we have extended the studies on our previously characterized cocaine antagonist, JHW 007 (N-n-butyl-4, 4-diF-benztropine), and discovered that JHW 007 and structurally related benztropine analogues were not self administered, in rats and indeed could selectively attenuate self administration of cocaine while having no effect on food administration. These studies further demonstrate the lack of abuse liability for these agents and strengthen their potential for development as therapeutics. Further evaluation of these and other N-substituted benztropines 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. Further investigation has shown that several additional analogues of JHW 007 show similar profiles in vivo and have been identified as lead candidates for development as medications to treat cocaine abuse, as well as attention deficit hyperactivity disorder (ADHD). Recent studies using site-directed mutagenesis have revealed differences in binding domains between the benztropines, cocaine and other structurally diverse dopamine uptake inhibitors. Interestingly, experimental evidence using the DAT inhibitors cocaine, WIN 35,428, and several benztropine analogues and comparing them to the substrates dopamine and MDMA has provided evidence, at the molecular level, of binding interaction differences that correlate with their distinctive behavioral profiles. Of note, cocaine binds to an outward facing conformation of the DAT, whereas the benztropines as well as the substrates, prefer an inward facing closed conformation. These conformational studies provide evidence at the molecular level that the atypical dopamine uptake inhibitors are indeed functioning differently than cocaine at the DAT and this is correlated with their distinct behavioral profiles. Our newest synthetic studies are focused on novel 2-substituted benztropine and benztropinamine analogues that incorporate oxazole functionality into the parent structure. 
Several of these compounds demonstrate high affinity and selectivity for the DAT and are currently being tested in the Y156F and Y335A mutants to determine preference for the outward or inward facing conformations of the DAT. Behavioral evaluation of selected analogues will follow. In addition, modafinil binds to the DAT and is currently used clinically for the treatment of sleep disorders. Recently, modafinil has been evaluated as a potential medication to treat methamphetamine and cocaine abuse and is also being used off-label for the treatment of ADHD. As modafinil has structural and pharmacological features that resemble the benztropines, we have embarked on a SAR study to further characterize these compounds at DAT, NET and SERT and to explore novel compounds with improved water solubility. The first series of compounds demonstrated a unique SAR profile and efforts to separate enantiomers of selected analogues are underway. In addition, computational studies support empirical studies in the mutant DATs that suggest modafinil prefers an inward facing conformation of the DAT. However, it is binding in a way that is unique to both cocaine and the benztropines.
 Further characterization of modafinil and its novel analogues will help elucidate the mechanisms underlying modafinils therapeutic action and thus far support the further development of the R-enantiomer as a psychostimulant medication strategy. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of fluorescent-derivatives of our tropane based DAT inhibitors. One such compound, JHC 1-064, has been used to characterize the trafficking and cellular distribution of DAT in living neuronal cells. Indeed, recent experiments in living neurons with JHC 1-064 have provided data that challenge mechanistic dogma for transporter translocation, as determined in DAT transfected heterologous cells, which is one area of ongoing research with these agents. Further, JHC 1-064 binds with high affinity to the serotonin transporter (SERT), as well, and we are conducting analogous studies, first in cell lines to study trafficking and cellular distribution of SERT. This project has led to a new project in which we have designed and synthesized novel analogues of the SERT inhibitor and antidepressant, citalopram. We have recently prepared a fluorescent citalopram analogue that is currently being used to visualize SERT in living cells. Citalopram is a racemic mixture of (+)- and (-) enantiomers, of which the latter (S-(+)-citalopram) is the more active antidepressant and currently marketed as Escitalopram. Previously obtained data suggest that the S-(+)-citalopram also binds to an allosteric site (S2) that can affect binding at the primary S1 site. To further explore this S2 site, we have designed and synthesized several series of citalopram analogues and separated several sets of enantiomers to further characterize and compare binding profiles at both the SERT and the other monoamine transporters, with the parent ligand. Our goal is to ultimately use these citalopram analogues to characterize the role of this putative allosteric S2 site. Collectively, these novel SERT compounds are being used to further elucidate structure and function of the SERT by characterizing primary and secondary binding domains that are related in an as of yet undetermined way to behavior.

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和强化作用的主要机制。为了进一步了解可卡因滥用的分子机制，结构-功能研究已在分子水平上定向表征DAT蛋白。3- α -（二苯甲氧基）tropane （benztropine）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，对许多苯托品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，苯托品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，也不能明显地在大鼠、恒河猴或松鼠猴身上自我施用。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。因此，我们将这类化合物描述为非典型多巴胺摄取抑制剂。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）。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 -取代苯托品类似物相比，这些化合物很容易穿透血脑屏障，但与可卡因相比，它们通常具有较慢的起效时间和较长的作用时间。这是一个适合作为药物治疗发展的特征，可能与他们缺乏类似可卡因的行为特征直接相关。目前正在进一步研究这类化合物的相关结构、药理作用和PD，并开发这些药物作为潜在的可卡因滥用治疗药物。在这方面，我们扩展了我们之前表征的可卡因拮抗剂JHW 007 （n- n-丁基- 4,4 -二f -苯托品）的研究，发现JHW 007和结构相关的苯托品类似物在大鼠中不是自我给药的，并且确实可以选择性地减弱可卡因的自我给药，而对食物给药没有影响。这些研究进一步证明了这些药物缺乏滥用责任，并加强了它们作为治疗药物发展的潜力。使用微透析对这些和其他n -取代苯托品进行进一步评估，使我们能够将体内细胞外多巴胺增加的速率和水平与这些化合物与DAT的结合联系起来。进一步的研究表明，JHW 007的其他几种类似物在体内表现出类似的特征，并已被确定为治疗可卡因滥用和注意缺陷多动障碍（ADHD）的主要候选药物。最近使用定点诱变的研究揭示了苯托品、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，使用DAT抑制剂可卡因、WIN 35,428和几种苯托品类似物的实验证据，并将它们与底物多巴胺和MDMA进行比较，在分子水平上提供了与它们独特的行为特征相关的结合相互作用差异的证据。值得注意的是，可卡因与DAT的外向构象结合，而苯托品和底物则倾向于内向封闭构象。这些构象研究在分子水平上提供了证据，证明非典型多巴胺摄取抑制剂在DAT中的功能确实不同于可卡因，这与它们独特的行为特征有关。我们最新的合成研究集中在新的2-取代苯托品和苯托品胺类似物上，这些类似物将恶唑的功能纳入母体结构。&#8232；其中一些化合物对DAT表现出高亲和力和选择性，目前正在Y156F和Y335A突变体中进行测试，以确定对DAT的外向或内向构象的偏好。随后将对选定的类似物进行行为评估。此外，莫达非尼与DAT结合，目前临床上用于治疗睡眠障碍。最近，莫达非尼被评估为治疗甲基苯丙胺和可卡因滥用的潜在药物，也被用于治疗多动症。由于莫达非尼具有与苯托品相似的结构和药理特征，我们已经开始了一项SAR研究，以进一步在DAT、NET和SERT上表征这些化合物，并探索具有改善水溶性的新化合物。第一个系列的化合物表现出独特的SAR特征，并且正在努力分离选定的类似物的对映体。此外，计算研究支持突变DAT的经验研究，表明莫达非尼更倾向于DAT的内向构象。然而，它以一种独特的方式结合可卡因和苯托品。&#8232；对莫达非尼及其新型类似物的进一步表征将有助于阐明莫达非尼治疗作用的机制，从而支持r -对构象体作为精神兴奋剂药物策略的进一步发展。除了开发用于体内研究的药物外，我们还以tropane基DAT抑制剂的荧光衍生物的形式合成了许多重要的分子工具。一种这样的化合物，JHC 1-064，已经被用来表征DAT在活神经元细胞中的运输和细胞分布。事实上，最近在JHC 1-064活神经元中进行的实验提供了挑战转运体易位机制的数据，正如在DAT转染的异源细胞中确定的那样，这是这些药物正在进行的研究的一个领域。此外，JHC 1-064也与血清素转运体（SERT）具有高亲和力结合，我们正在进行类似的研究，首先在细胞系中研究SERT的运输和细胞分布。这个项目导致了一个新的项目，在这个项目中，我们设计和合成了SERT抑制剂和抗抑郁药西酞普兰的新型类似物。我们最近制备了一种荧光西酞普兰类似物，目前用于在活细胞中可视化SERT。西酞普兰是(+)-和（-）对映异构体的外消旋混合物，后者（S-(+)-西酞普兰）是更有效的抗抑郁药，目前以艾司西酞普兰的名字上市。先前获得的数据表明，S-(+)-citalopram还可以结合到一个变构位点（S2），从而影响主要S1位点的结合。为了进一步探索这个S2位点，我们设计并合成了几个系列的西酞普兰类似物，并分离了几组对映体，以进一步表征和比较SERT和其他单胺转运体与母体配体的结合谱。我们的目标是最终使用这些西酞普兰类似物来表征这个假定的变构S2位点的作用。总的来说，这些新的SERT化合物正被用于进一步阐明SERT的结构和功能，通过表征以尚未确定的方式与行为相关的初级和次级结合域。