Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.
Click Chemistry-Enabled Conjugation Strategy for Producing Dibenzodiazepinone-Type Fluorescent Probes To Target M2 Acetylcholine Receptors.
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DOI：
10.1021/acs.bioconjchem.2c00446
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发表时间：
2022-11
影响因子：
4.7
通讯作者：
Hongrong Yang;N. Micovic;James R. Monaghan;H. Clark
中科院分区：
文献类型：
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作者：
Hongrong Yang;N. Micovic;James R. Monaghan;H. Clark
荧光标记受体靶向化合物的发展为研究和表征配体-受体相互作用提供了一种强大的药理学工具。尽管在开发M胆碱型乙酰胆碱受体(MAChRs)亚型特异性拮抗剂方面取得了重大进展，但关于可用于点击化学的拮抗剂的报道较少。在这里，我们基于先前报道的与2型mAChR(M2R)具有高结合亲和力的二苯二氮卓酮(DiBA)支架，通过点击化学方法设计并合成了一种适合于探针连接的拮抗剂，即二苯二氮卓酮(DiBA)-炔。为了证明二巴基炔作为生物偶联物的构筑基团的多功能性，我们将二巴基炔与氰基-5-荧光团(Cy5)和聚乙二醇(PEG)生物分子组装在一起，得到了荧光的DiBA拮抗剂(DiBA-Cy5)和荧光DiBA的聚乙二醇衍生物。流式细胞仪分析表明，DiBA-Cy5与M2R具有较高的结合亲和力(Kd=1.80 nM)，比M1R高两个数量级。共聚焦显微镜成像证实，荧光DIBA聚乙二醇衍生物与M2R(KD≤4 NM)具有很强的结合能力。此外，DiBA-Cy5可以作为其他mAChR配体的受体-配体竞争结合分析中的荧光配体，这是传统的基于放射性配体的分析的一个有吸引力的替代方法。DIBA-Cy5与正构拮抗剂阿托品/变构调节剂LY2119620的竞争结合模式表明，DiBA型拮抗剂与M2R呈双立体结合模式。最后，我们展示了DIBA-Cy5对M2R受体在小鼠心脏窦房结的直接染色。可点击的DiBA拮抗剂对广泛的荧光团和生物分子的适应性可以促进其在各种生物医学应用中的应用，例如以M2R为受体靶点筛选化合物的结合分析。
The development of fluorescently labeled receptor-targeting compounds represents a powerful pharmacological tool to study and characterize ligand-receptor interactions. Despite significant advances in developing sub-type-specific antagonists for muscarinic acetylcholine receptors (mAChRs), reports on antagonists feasible for click chemistry are less common. Here, we designed and synthesized an antagonist suitable for probe attachment through click chemistry, namely, dibenzodiazepinone (DIBA)-alkyne, based on a previously reported DIBA scaffold with a high binding affinity to type-2 mAChR (M2R). To demonstrate the versatility of DIBA-alkyne as a building block for bioconjugates, we assembled DIBA-alkyne with Cyanine5 fluorophores (Cy5) and polyethylene glycol (PEG) biomolecules to obtain fluorescent DIBA antagonist (DIBA-Cy5) and fluorescent DIBA PEG derivatives. Flow cytometric analysis showed that DIBA-Cy5 possessed a high binding affinity to M2R (Kd = 1.80 nM), a two-order magnitude higher binding affinity than M1R. Fluorescent DIBA PEG derivatives maintained a potent binding to the M2R (Kd ≤ 4 nM), confirmed by confocal microscopic imaging. Additionally, DIBA-Cy5 can serve as a fluorescent ligand in the receptor-ligand competitive binding assay for other mAChR ligands, an attractive alternative to the traditional radioligand-based assay. The competitive binding mode between DIBA-Cy5 and orthosteric antagonist atropine/allosteric modulator LY2119620 indicated a dualsteric binding mode of the DIBA-type antagonist to M2R. Lastly, we demonstrated the direct staining of DIBA-Cy5 to M2R receptors in the sinoatrial node of a mouse heart. The adaptability of the clickable DIBA antagonist to a wide range of fluorophores and biomolecules can facilitate its use in various biomedical applications such as binding assays that screen compounds for M2R as the receptor target.