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Target immobilization on glass microfiber membranes as a label-free strategy for hit identification

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Abstract

The discovery of novel chemical entities targeting G protein-coupled receptors (GPCRs) is usually guided by their receptor affinity. However, traditional affinity assay methods and hit identification procedures are usually laborious and expensive. In this work, the type-2 vasopressin receptor (V2R) was chosen as a prototypical GPCR. Membrane fragments from cells highly expressing SNAP-V2R were immobilized on the surface of a glass microfiber (GMF) coated with O6-benzylguanine (BG). This was achieved by transferring the benzyl group of BG to the active site of the SNAP-tag through a nucleophilic substitution reaction. As a result, a biofilm called SNAP-V2R@GMF-BG was produced that showed good specificity and stability. The adsorption ratio for each V2R ligand treated with SNAP-V2R@GMF-BG was determined by HPLC and exhibited a good linear correlation with the Ki value determined by displacement assays. Furthermore, a Ki prediction assay was performed by comparing the data with that generated by a homogeneous time-resolved fluorescence (HTRF) assay. SNAP-V2R@GMF-BG was also used to screen hit compounds from natural products. After SNAP-V2R@GMF-BG was incubated with the total extract, the ligand that binds to V2R could be separated and subjected to LC‒MS analysis for identification. Baicalein was screened from Clerodendranthus spicatus and verified as a potential V2R antagonist. This V2R-immobilized GMF platform can help determine the affinity of V2R-binding hit compounds and screen the compounds efficiently and accurately.

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Abbreviations

AVP:

Arginine vasopressin

GPCR:

G protein-coupled receptor

TR-FRET:

Time-resolved fluorescence resonance energy transfer

V2R:

Type-2 vasopressin receptor

GMF:

Glass microfiber filters

BG:

O6-Benzylguanine

CS:

Clerodendran thus spicatus

HTRF:

Homogeneous time-resolved fluorescence

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Acknowledgements

The authors thank Biotree Biomedical Technology Co., Ltd., for technical assistance with HRMS analysis.

Funding

This project was financially supported by the National Natural Science Foundation of China (22077110, 22377103, 8234869), the Natural Science Foundation of Jiangsu Province (BK20200106), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (20KJA350001, 21KJB350025), and the project of scientific and technological development of traditional Chinese medicine in Jiangsu province (MS2021106). This project was partly performed at the National Demonstration Center for Experimental Basic Medical Science Education (Xuzhou Medical University) and funded by the Innovation and Entrepreneurship Training Program for College Students in Jiangsu Province (grant number 202210313024Z).

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  1. Yinan Wang and Yan He contributed equally in the making of this manuscript.

Authors and Affiliations

  1. Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221004, Jiangsu, China

    Yinan Wang, Yan He, Xiaojiao Ye, Yixiao Zhang, Xiuxiu Huang, Hongli Liu, Wenqing Dong, Dongzhi Yang & Dong Guo

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Contributions

Yinan Wang: original draft. Yan He: data curation. Xiaojiao Ye: editing and visualization. Yixiao Zhang: methodology. Xiuxiu Huang: validation. Hongli Liu: software. Wenqing Dong: visualization. Dongzhi Yang: supervision. Dong Guo: writing — review and editing.

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Correspondence to Dongzhi Yang or Dong Guo.

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Wang, Y., He, Y., Ye, X. et al. Target immobilization on glass microfiber membranes as a label-free strategy for hit identification. Anal Bioanal Chem 415, 6743–6755 (2023). https://doi.org/10.1007/s00216-023-04951-w

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