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。值得注意的是，可卡因与面向外的DAT构象结合，而我们的非典型DAT抑制剂更倾向于面向内的闭塞构象。先前的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。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，但效果有限，也被用于治疗多动症。到目前为止，这些新颖的类似物展示了独特的SAR剖面。对莫达非尼模板的修饰导致分子具有高亲和力（比母体药物高1000倍）并选择性地与DAT结合。此外，计算研究支持突变DAT的实验，表明莫达非尼更倾向于DAT的更闭塞的构象，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、药代动力学和行为学分析确定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RDS03-94和RDS4-010，正在可卡因滥用啮齿动物模型中进行评估。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强化作用，而不显着影响伏隔核中的多巴胺水平，通过微透析和快速扫描循环伏安法以及电生理学测量。我们已经证明，这种结构模板的非常细微的差异可以将非典型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）。分子水平上这些细微的结构变化可以深刻地改变这些分子的行为特征，而这些在原子水平上的见解，已经导致了治疗精神兴奋剂使用障碍的潜在药物疗法的新药物设计。除了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，减轻与人类DAT基因突变相关的严重致残性运动障碍。去甲博格碱最近被描述为一种药物伴侣，能够挽救突变的DAT，这些DAT无法在内质网中正常折叠，因此无法到达膜，使患者的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，以确定可能挽救这些突变DAT的铅分子，并提供改善DAT功能和改善与这些疾病相关的症状的机会。我们已经确定了一系列解构的伊博格碱类似物，在我们的实验室合成，表现出超过去甲博格碱的DAT和SERT药物伴随活性。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。

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.