Novel Probes for the Monoamine Transporters

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。值得注意的是，可卡因与DAT的外向构象结合，而我们的非典型DAT抑制剂更喜欢内向封闭构象。以前的构象研究提供的证据，在分子水平上，非典型多巴胺摄取抑制剂确实是功能不同的可卡因在DAT，这是与他们不同的行为概况。最近，我们把注意力集中在莫达非尼上，它与DAT结合，目前临床上用于治疗睡眠障碍。莫达非尼已被评估为治疗甲基苯丙胺和可卡因滥用的潜在药物，但成功有限，也被用于治疗ADHD。到目前为止，这些新的类似物表现出独特的SAR曲线。对莫达非尼模板的修饰已经产生了具有高亲和力（比母体药物高>1000倍）和选择性结合DAT的分子。此外，计算研究支持突变DAT中的实验，该实验表明莫达非尼更喜欢DAT的封闭构象，更像我们以前和广泛发表的苯扎托品类似物而不是可卡因。代谢、药代动力学和行为分析确定JJC 8 -091为进一步开发的先导化合物。最近，在可卡因滥用的啮齿动物模型中对包括RDS 03 -94和RDS 4 -010在内的几种新的类似物进行了评价。 机制研究正在进行中，以阐明这些新的DAT抑制剂如何阻断精神兴奋剂的增强作用，而不显着影响伏隔核中的多巴胺水平，如通过微透析和快速扫描循环伏安法以及电生理学测量的。 我们已经证明，这种结构模板中非常细微的差异可以转化非典型DAT抑制剂（例如，JJC 8 -091）转化为更典型的可卡因样分子（例如，JJC 8 -088）。分子水平上的这些微妙结构变化可以深刻地改变这些分子的行为特征，而原子水平上的这些见解已经为治疗精神兴奋剂使用障碍的潜在药物疗法带来了新型药物设计。除了对精神兴奋剂使用障碍的药物潜力外，非典型DAT抑制剂还可以作为药物伴侣，其可以减轻与DAT中的人类基因突变相关的严重致残性运动障碍。Noriboclavin最近被描述为药物伴侣，能够拯救不能在内质网中正确折叠的突变DAT，因此永远不会到达膜，使患者DAT功能差，导致运动和神经精神障碍。 因此，我们已经开始了筛选和合成项目，以确定可能挽救这些突变DAT的先导分子，并提供改善DAT功能和改善与这些疾病相关的症状的机会。我们已经鉴定了一系列在我们实验室合成的解构的iboboxin类似物，其表现出超过noriboboxin的DAT和SERT药物伴侣活性。进一步的分子药理学和计算建模研究正在进行中，以促进我们对这些作用的机制基础的理解，以及体内研究。 除了开发用于体内研究的药物外，我们还合成了许多针对单胺转运蛋白的重要生物缀合物分子工具。我们的基于托烷的荧光配体JHC 1 -064已在许多实验室中用于表征SERT，NET和DAT在活神经元细胞中的运输和细胞分布。最近，我们已经设计了新的荧光配体，使用定制的荧光团适用于现场超分辨率成像。修饰托烷药效团和荧光团之间的连接体以及用超亮JaneliaFluor（JF）荧光团取代JHC 1 -064的罗丹明，产生了新的荧光配体DG 3 -80和DG 4 -91，它们目前被用于使用超分辨率显微镜观察DAT。此外，还合成了基于NET抑制剂他洛普伦、尼索西汀和哌醋甲酯的新型荧光配体，其中一种尼索西汀类似物显示出优异的NET选择性，并且发现哌醋甲酯类似物适用于基于FRET的突触蛋白-III结合测定。

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. Of note, cocaine binds to an outward facing conformation of the DAT, whereas the our atypical DAT inhibitors prefer an inward facing occluded conformation. Previous conformational studies provided evidence, at the molecular level, that the atypical dopamine uptake inhibitors are indeed functioning differently than cocaine at the DAT and this is related to their distinct behavioral profiles. More recently we have focused attention on modafinil, which binds to the DAT and is currently used clinically for the treatment of sleep disorders. Modafinil has been evaluated as a potential medication to treat methamphetamine and cocaine abuse, with limited success, and is also being used off-label for the treatment of ADHD. Thus far these novel analogs demonstrate a unique SAR profile. Modifications to the modafinil template have resulted in molecules with high affinity (>1000-fold higher than the parent drug) and selective binding to the DAT. In addition, computational studies support experiments in the mutant DATs that suggest modafinil prefers a more occluded conformation of the DAT, more like our previously and extensively published benztropine analogs than cocaine. Metabolism, pharmacokinetic and behavioral analyses identified JJC8-091 as lead compounds for further development. More recently several new analogues, including RDS03-94 and RDS4-010 are being evaluated in rodent models of cocaine abuse. Mechanistic studies are underway to elucidate how these novel DAT inhibitors block the reinforcing effects of psychostimulant without significantly affecting dopamine levels in the Nucleus Accumbens, as measured by microdialysis and fast scanning cyclic voltammetry, as well as electrophysiology. We have demonstrated that very subtle differences in this structural template can convert an atypical DAT inhibitor (e.g., JJC8-091) into a more typical cocaine-like molecule (e.g., JJC8-088). These subtle structural changes at the molecular level can profoundly change the behavioral profile of these molecules and these insights at the atomistic level, have led to novel drug design for potential pharmacotherapies to treat psychostimulant use disorders. In addition to medication potential for psychostimulant use disorders, atypical DAT inhibitors may also serve as pharmacochaperones that may mitigate the severely disabling motor disorders associated with human genetic mutations in DAT. Noribogaine has recently been described as a pharmacochaperone, capable of rescuing mutant DATs that are unable to fold properly in the endoplasmic reticulum, so never make it to the membrane, leaving the patient with poor DAT function that results in movement and neuropsychiatric disorders. Hence we have embarked on a screening and synthesis project to identify lead molecules that may rescue these mutant DATs and provide the opportunity to improve DAT function and ameliorate symptoms associated with these disorders. We have identified a series of deconstructed ibogaine analogues, synthesized in our lab, that exhibit DAT and SERT pharmacochaperoning activities that surpass those of noribogaine. Further molecular pharmacology and computational modeling studies are underway to advance our understanding of the mechanistic underpinnings of these actions, as well as in vivo studies. In addition to developing agents for in vivo studies, we have also synthesized a number of important bioconjugate molecular tools directed toward the monoamine transporters. Our fluorescent tropane-based ligand, JHC1-064, has been used in many labs to characterize the trafficking and cellular distribution of SERT, NET and DAT in living neuronal cells. More recently, we have designed of novel fluorescent ligands, using customized fluorophores suitable for live super resolution imaging. Modification of the linker between the tropane pharmacophore and the fluorophore as well as replacing the rhodamine of JHC1-064 with super bright JaneliaFluor (JF) fluorophores has resulted in the novel fluorescent ligands DG3-80 and DG4-91 that are currently being used to visualize DAT using super resolution microscopy. Moreover, novel fluorescent ligands based on the NET inhibitors talopram, nisoxetine and methylphenidate were also synthesized, with one nisoxetine analogue showing excellent NET selectivity and a methylphenidate analog was discovered to be suitable for FRET-based assays for Synapsin-III binding.