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使用障碍的分子机制，结构-功能研究已经指向在分子水平上表征DAT蛋白。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了证据，在分子水平上，非典型多巴胺摄取抑制剂确实在DAT上与可卡因不同，这与它们不同的行为特征有关。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评价了几种新的类似物，包括RDS 03 -94和RDS 4 -010。 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）。这些在分子水平上的细微结构变化可以深刻地改变这些分子的行为特征，并且这些在原子水平上的见解已经导致了用于治疗精神兴奋剂使用障碍（PSUD）的潜在药物疗法的新型药物设计。最近，我们将药物设计的重点放在改善代谢稳定性和降低hERG通道活性（心脏毒性的预测因子）上。这项工作正在产生新的小分子，这些小分子既具有所需的药理学特征，又具有药物样特性，这将提高它们转化为临床的机会。除了PSUD的药物潜力外，非典型DAT抑制剂还可以作为药物伴侣，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。诺利博韦最近被描述为药物伴侣，能够拯救无法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伊波替尼类似物，其表现出超过诺波替尼的DAT和SERT药物伴侣活性，我们目前的领先者是DG 4 -69。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，MFZ 9 -18，一种俄勒冈州绿色标记的托烷基荧光团，最近被用于可视化恒河猴脑组织切片中的多巴胺能轴突，使得能够进行膜片钳电生理学实验。我们现在已经合成了一系列新的荧光配体的基础上，我们的莫达非尼类似物，JJC 8 -091和JJC 8 -088，使用改性的连接器和荧光团添加到我们的工具箱的DAT的荧光工具。此外，新的荧光配体的NET抑制剂他洛普伦，nisoxetine和哌甲酯的基础上也合成了一个nisoxetine类似物表现出良好的NET选择性。发现哌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 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.