Swiss Federal Institute of Technology Lausanne (EPFL).
Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School.
J Vis Exp. 2023 Jun 2(196). doi: 10.3791/64846.
Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.
反应性代谢物和相关的亲电药物是最难研究的小分子之一。传统的方法来解构这种分子的作用模式（MOA）利用过量的特定反应性物质对实验标本进行批量处理。在这种方法中，亲电试剂的高反应性以时间和上下文依赖的方式对蛋白质组进行非选择性标记；氧化还原敏感的蛋白质和过程也可以被间接且经常不可逆地影响。在这种无数潜在目标和间接次要效应的背景下，将表型与特定的靶标结合仍然是一项复杂的任务。针对反应性亲电试剂和氧化剂的斑马鱼（Z-REX）——一种适应于幼鱼斑马鱼的按需反应性亲电试剂输送平台——旨在将亲电试剂递送到感兴趣的特定蛋白质（POI），而不会对未受干扰的活体胚胎鱼造成影响。该技术的关键特征包括低侵入性、剂量、化学型和时空控制的精确亲电试剂输送。因此，结合独特的控制套件，该技术可以避免非靶向效应和系统性毒性，否则在对动物进行不受控制的亲电试剂和多效亲电药物批量暴露时会观察到这些效应。利用 Z-REX，研究人员可以在理解个体应激反应和信号输出如何因特定反应性配体与特定 POI 的结合而改变方面取得进展，这种理解是在完整活体动物的近生理条件下进行的。
