Discovery of a novel OGT inhibitor through high-throughput screening based on Homogeneous Time-Resolved Fluorescence (HTRF)

Xinyu Wu; Mingchen Wang; Yu Cao; Ying Xu; Ziqun Yang; Yiluan Ding; Jing Lu; Jie Zheng; Cheng Luo; Kehao Zhao; Shijie Chen

doi:10.1016/j.bioorg.2023.106726

Discovery of a novel OGT inhibitor through high-throughput screening based on Homogeneous Time-Resolved Fluorescence (HTRF)

Bioorg Chem. 2023 Oct:139:106726. doi: 10.1016/j.bioorg.2023.106726. Epub 2023 Jul 8.

Authors

Xinyu Wu¹, Mingchen Wang², Yu Cao³, Ying Xu⁴, Ziqun Yang⁵, Yiluan Ding⁶, Jing Lu⁷, Jie Zheng⁵, Cheng Luo⁸, Kehao Zhao⁹, Shijie Chen¹⁰

Affiliations

¹ School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China; Drug Discovery and Design Center, The Center for Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
² Drug Discovery and Design Center, The Center for Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; School of Life Science and Technology, ShanghaiTech University, 100 Haike Road, Shanghai 201210, China.
³ Drug Discovery and Design Center, The Center for Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu, China.
⁴ Drug Discovery and Design Center, The Center for Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; China Pharmaceutical University, Nanjing 210009, China.
⁵ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China; Center of Immunological Diseases, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
⁶ University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China; Analytical Research Center for Organic and Biological Molecules, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.
⁷ School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China.
⁸ Drug Discovery and Design Center, The Center for Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China; School of Pharmaceutical Science and Technology, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou 310024, China; School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu, China.
⁹ School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai 264005, China. Electronic address: kehaozhao@gmail.com.
¹⁰ Drug Discovery and Design Center, The Center for Chemical Biology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, China. Electronic address: shijiechen@simm.ac.cn.

PMID: 37451145
DOI: 10.1016/j.bioorg.2023.106726

Abstract

O-GlcNAcylation is a specific type of post-translational glycosylation modification, which is regulated by two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA). Aberrant overexpression of OGT is associated with the development of many solid tumors. In this study, we have developed and optimized a sensitive Homogeneous Time-Resolved Fluorescence (HTRF) assay then identified a novel OGT inhibitor CDDO (also called Bardoxolone) through a high-throughput screening (HTS) based on HTRF assay. Further characterization suggested that CDDO is an effective OGT inhibitor with an IC₅₀ value of 6.56 ± 1.69 μM. CPMG-NMR analysis confirmed that CDDO is a direct binder of OGT with a binding affinity (K_d) of approximately 1.7 μM determined by the MST analysis. Moreover, HDX-MS analysis indicated that CDDO binds to the TPR domain and N-Terminal domain of OGT, which was further confirmed by the enzymatic competition experiments as the binding of CDDO to OGT was not affected by the catalytic site binding inhibitor OSMI-4. Our docking modeling analysis further predicted the possible interactions between CDDO and OGT, providing informative molecular basis for further optimization of the inhibitor in the future. Together, our results suggested CDDO is a new inhibitor of OGT with a distinct binding pocket from the reported OGT inhibitors. Our work paved a new direction for developing OGT inhibitors driven by novel mechanisms.

Keywords: High-throughput screening; Inhibitor; O-GlcNAc transferase; O-GlcNAcylation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Glycosylation
High-Throughput Screening Assays*
Protein Processing, Post-Translational*

Substances

O-GlcNAc transferase
bardoxolone