Novel Probes for the Monoamine Transporters

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使用障碍的分子机制，在分子水平上对DAT蛋白进行了结构-功能研究。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抑制剂更喜欢内向的闭塞构象。以前的构象研究在分子水平上提供了证据，证明非典型的多巴胺摄取抑制剂在DAT中的作用确实不同于可卡因，这与它们不同的行为特征有关。最近，我们将注意力集中在莫达非尼上，它与DAT结合，目前正被临床用于治疗睡眠障碍。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，但成效有限，也被用于治疗ADHD。到目前为止，这些新颖的类比展示了独特的合成孔径雷达图谱。对莫达非尼模板的修饰已经产生了具有高亲和力(比母药高1000倍)并选择性地与DAT结合的分子。此外，计算研究支持在突变的DAT中的实验，表明莫达非尼更喜欢DAT的更封闭的构象，更像我们之前广泛发表的苯托品类似物，而不是可卡因。代谢、药代动力学和行为分析确定JJC8-091为进一步开发的先导化合物。最近，一些新的类似物，包括RDS 03-94和RDS 4-010，已经在可卡因滥用的啮齿动物模型中进行了评估。机制研究正在进行中，以阐明这些新型DAT抑制剂如何在不显著影响伏核中多巴胺水平的情况下阻断精神刺激剂的增强效应，如微透析和快速扫描循环伏安法以及电生理。我们已经证明，这个结构模板中非常细微的差异可以将非典型的DAT抑制剂(例如JJC8-091)转化为更典型的可卡因样分子(例如JJC8-088)。分子水平上的这些微妙的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导致了治疗精神刺激剂使用障碍(PSUD)的潜在药物疗法的新药物设计。最近，我们的药物设计集中于改善代谢稳定性和降低HERG通道活性，HERG通道活性是心脏毒性的预测指标。这项工作正在导致新的小分子，既具有所需的药理学特征，又具有类似药物的特性，将提高它们转化到临床的机会。除了治疗PSUD的药物潜力外，非典型的DAT抑制剂也可以作为药物伴侣，缓解与人类DAT基因突变相关的严重致残性运动障碍。Noribogaine最近被描述为一种药物，能够拯救突变的DAT，这些突变的DAT无法在内质网中正确折叠，因此永远不能进入膜，导致患者DAT功能低下，导致运动和神经精神障碍。因此，我们开始了一项筛选和合成项目，以确定可能拯救这些突变的DAT的先导分子，并提供机会来改善DAT功能，改善与这些疾病相关的症状。我们已经鉴定了一系列在我们实验室合成的解构的ibogaine类似物，它们表现出超过noribogaine的DAT和SERT药理作用，我们目前的领先是DG4-69。进一步的分子药理学和计算模型研究正在进行中，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试剂外，我们还合成了一些针对单胺转运体的重要生物偶联分子工具。我们的荧光托烷配体JHC1-064已被许多实验室用来表征SERT、Net和DAT在活的神经细胞中的运输和细胞分布。最近，我们设计了新的荧光配体，使用适合现场超分辨率成像的定制荧光团。对托烷药效团和荧光团之间的连接体进行修饰，并用超明亮的珍妮亚荧光(JF)荧光团取代JHC1-064的罗丹明，得到了新的荧光配体DG3-80和DG4-91，目前正在使用超分辨率显微镜对DAT进行可视化。此外，俄勒冈州绿色标记的基于托烷的荧光团MFZ9-18最近被用于显示猕猴脑组织切片中的多巴胺能轴突，使膜片钳电生理学实验得以进行。我们现在已经合成了基于我们的莫达非尼类似物JJC8-091和JJC8-088的一系列新的荧光配体，使用修饰的连接物和荧光团来添加到我们的DAT荧光工具工具箱中。此外，还合成了基于Net抑制剂他洛普兰、尼索西汀和哌酸甲酯的新型荧光配体，其中一个Nisoxetine类似物表现出良好的Net选择性。研究发现，哌醋甲酯类似物适用于基于FRET的突触素-III结合分析。

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 use disorder, 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 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 have been 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 (PSUD). Recently we have focused our drug design toward improving both metabolic stability and reducing hERG channel activity, a predictor of cardiotoxicity. This work is leading to new small molecules that have both the desired pharmacological profiles as well as drug-like properties that will improve their chances of translation to the clinic. In addition to medication potential for PSUD, 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 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, with our current lead being DG4-69. 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. In addition, MFZ9-18, an Oregon Green-labeled tropane-based fluorophore, was recently used to visualize dopaminergic axons in rhesus macaque brain tissue slices enabling patch clamp electrophysiology experiments to be performed. We have now synthesized a new series of fluorescent ligands based on our modafinil analogues, JJC8-091 and JJC8-088, using modified linkers and fluorophores to add to our toolbox of fluorescent tools for DAT. Moreover, novel fluorescent ligands based on the NET inhibitors talopram, nisoxetine and methylphenidate were also synthesized, with one nisoxetine analogue showing excellent NET selectivity. The methylphenidate analog was discovered to be suitable for FRET-based assays for Synapsin-III binding.