Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish.,ACS Chemical Neuroscience

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Combining Neurobehavioral Analysis and In Vivo Photoaffinity Labeling to Understand Protein Targets of Methamphetamine in Casper Zebrafish.
ACS Chemical Neuroscience ( IF 4.1 ) Pub Date : 2020-07-30 , DOI: 10.1021/acschemneuro.0c00416
Ethel Tackie-Yarboi ₁ , Alexander Wisner ₂ , Austin Horton ₁ , Tue Q T Chau ₁ , James Reigle _{3,

4} , Adam J Funk ₅ , Robert E McCullumsmith _{5,

6} , Frank S Hall ₂ , Frederick E Williams ₂ , Isaac T Schiefer _{1,

7}

Affiliation

Photoaffinity labeling (PAL) remains one of the most widely utilized methods of determining protein targets of drugs. Although useful, the scope of this technique has been limited to in vitro applications because of the inability of UV light to penetrate whole organisms. Herein, pigment-free Casper zebrafish were employed to allow in vivo PAL. A methamphetamine-related phenethylamine PAL probe, designated here as 2, demonstrated dose-dependent effects on behavior similar to methamphetamine and permitted concentration-dependent labeling of protein binding partners. Click chemistry was used to analyze binding partners via fluoroimaging. Conjugation to a biotin permitted streptavidin pull-down and proteomic analysis to define direct binding partners of the methamphetamine probe. Bioinformatic analysis revealed the probe was chiefly bound to proteins involved in phagocytosis and mitochondrial function. Future applications of this experimental paradigm combining examination of drug–protein binding interactions alongside neurobehavioral readouts via in vivo PAL will significantly enhance our understanding of drug targets, mechanism(s) of action, and toxicity/lethality.

中文翻译：

结合神经行为分析和体内光亲和标记来了解 Casper 斑马鱼中甲基苯丙胺的蛋白质靶标。

光亲和标记 (PAL) 仍然是确定药物蛋白质靶标的最广泛使用的方法之一。尽管有用，但由于紫外线无法穿透整个生物体，因此该技术的范围仅限于体外应用。在此，无色素 Casper 斑马鱼被用于允许体内PAL。甲基苯丙胺相关苯乙胺 PAL 探针，此处指定为2，证明了对类似于甲基苯丙胺的行为的剂量依赖性影响，并允许对蛋白质结合伴侣进行浓度依赖性标记。点击化学用于通过荧光成像分析结合伴侣。与生物素结合允许链霉亲和素下拉和蛋白质组学分析，以确定甲基苯丙胺探针的直接结合伙伴。生物信息学分析显示，该探针主要与参与吞噬作用和线粒体功能的蛋白质结合。这种实验范式的未来应用将药物-蛋白质结合相互作用的检查与通过体内PAL 的神经行为读数相结合，将显着增强我们对药物靶点、作用机制和毒性/致死率的理解。

更新日期：2020-09-02

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