CETSA-MS-based target profiling of anti-aging natural compound quercetin

• Published in: European journal of medicinal chemistry 2024-03, Vol.267, p.116203-116203, Article 116203
• Main Authors: Bai, Lin, Deng, Zhifen, Xu, Mengfei, Zhang, Zhehao, Guo, Guangyu, Xue, Xinli, Wang, Shaochi, Yang, Jinghua, Xia, Zongping
• Format: Article
• Language: English
• Subjects: CBR1; Cellular thermal shift assay; GSK3A; MAPK1; Quercetin; Thermal proteome profiling
• ISSN: 0223-5234; 1768-3254
• DOI: 10.1016/j.ejmech.2024.116203

Quercetin is widely distributed in nature and abundant in the human diet, which exhibits diverse biological activities and potential medical benefits. However, there remains a lack of comprehensive understanding about its cellular targets, impeding its in-depth mechanistic studies and clinical applications. This study aimed to profile protein targets of quercetin at the proteome level. A label-free CETSA-MS proteomics technique was employed for target enrichment and identification. The R package Inflect was used for melting curve fitting and target selection. D3Pocket and LiBiSco tools were used for binding pocket prediction and binding pocket analysis. Western blotting, molecular docking, site-directed mutagenesis and pull-down assays were used for target verification and validation. We curated a library of direct binding targets of quercetin in cells. This library comprises 37 proteins that show increased thermal stability upon quercetin binding and 33 proteins that display decreased thermal stability. Through Western blotting, molecular docking, site-directed mutagenesis and pull-down assays, we validated CBR1 and GSK3A from the stabilized protein group and MAPK1 from the destabilized group as direct binding targets of quercetin. Moreover, we characterized the shared chemical properties of the binding pockets of quercetin with targets. Our findings deepen our understanding of the proteins pivotal to the bioactivity of quercetin and lay the groundwork for further exploration into its mechanisms of action and potential clinical applications. [Display omitted] •Identification of quercetin's cellular protein targets using CETSA-MS.•Creation of a curated library with 37 stabilized and 33 destabilized proteins.•Identification of CBR1, GSK3A, and MAPK1 as direct quercetin binding targets.•Revelation of shared chemical properties in quercetin-target binding pockets.