Simple Fluorescence Labeling Method Enables Detection of Intracellular Distribution and Expression Level of Retinoid X Receptors

IF 3.5 3区医学 Q2 CHEMISTRY, MEDICINAL ACS Medicinal Chemistry Letters Pub Date : 2024-04-23 DOI:10.1021/acsmedchemlett.4c00033

Yukina Tanaka, Michiko Fujihara, Yuta Takamura, Mayu Kawasaki, Shogo Nakano, Makoto Makishima and Hiroki Kakuta*,

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Abstract

There is no straightforward method to visualize the intracellular distribution of nuclear receptors, such as retinoid X receptors (RXRs), which are trafficked between the cytosol and nucleus. Here, in order to develop a simple fluorescence labeling method for RXRs, we designed and synthesized compound 4, consisting of an RXR-selective antagonist, CBTF-EE (2), linked via an ether bond to the fluorophore nitrobenzoxadiazole (NBD). Compound 4 is nonfluorescent, but the ether bond (-O-NBD) reacts with biothiols such as cysteine and homocysteine to generate a thioether (-S-NBD), followed by intramolecular Smiles rearrangement with an amino group such as that of lysine to form a fluorescent secondary amine (-NH-NBD) adjacent to the binding site. Fluorescence microscopy of intact or RXR-overexpressing MCF-7 cells after incubation with 4 enabled us to visualize RXR expression as well as nuclear transfer of RXR induced by the agonist bexarotene (1).

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简单的荧光标记法可检测视黄醇 X 受体的细胞内分布和表达水平

视黄醇 X 受体（RXRs）等核受体在细胞质和细胞核之间流动，目前还没有直观的方法来观察它们在细胞内的分布。为了开发一种简单的 RXRs 荧光标记方法，我们设计并合成了化合物 4，它由 RXR 选择性拮抗剂 CBTF-EE (2) 通过醚键与荧光团硝基苯并噁二唑 (NBD) 连接而成。化合物 4 无荧光，但醚键（-O-NBD）与半胱氨酸和高半胱氨酸等生物硫醇反应生成硫醚（-S-NBD），然后与赖氨酸等氨基发生分子内 Smiles 重排，在结合位点附近形成荧光仲胺（-NH-NBD）。我们用荧光显微镜观察了与 4 培养后的完整或 RXR 表达的 MCF-7 细胞，从而观察到 RXR 的表达以及激动剂贝沙罗汀诱导的 RXR 核转移（1）。

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来源期刊

ACS Medicinal Chemistry Letters CHEMISTRY, MEDICINAL-

CiteScore

7.30

自引率

2.40%

发文量

328

审稿时长

1 months

期刊介绍： ACS Medicinal Chemistry Letters is interested in receiving manuscripts that discuss various aspects of medicinal chemistry. The journal will publish studies that pertain to a broad range of subject matter, including compound design and optimization, biological evaluation, drug delivery, imaging agents, and pharmacology of both small and large bioactive molecules. Specific areas include but are not limited to: Identification, synthesis, and optimization of lead biologically active molecules and drugs (small molecules and biologics) Biological characterization of new molecular entities in the context of drug discovery Computational, cheminformatics, and structural studies for the identification or SAR analysis of bioactive molecules, ligands and their targets, etc. Novel and improved methodologies, including radiation biochemistry, with broad application to medicinal chemistry Discovery technologies for biologically active molecules from both synthetic and natural (plant and other) sources Pharmacokinetic/pharmacodynamic studies that address mechanisms underlying drug disposition and response Pharmacogenetic and pharmacogenomic studies used to enhance drug design and the translation of medicinal chemistry into the clinic Mechanistic drug metabolism and regulation of metabolic enzyme gene expression Chemistry patents relevant to the medicinal chemistry field.