An In-Silico, In-Vitro and In-Vivo Combined Approach to Identify NMNATs as Potential Protein Targets of ProEGCG for Treatment of Endometriosis

Sze Wan Hung; Bo Liang; Yating Gao; Ruizhe Zhang; Zhouyurong Tan; Tao Zhang; Pui Wah Jacqueline Chung; Tak Hang Chan; Chi Chiu Wang

doi:10.3389/fphar.2021.714790

An In-Silico, In-Vitro and In-Vivo Combined Approach to Identify NMNATs as Potential Protein Targets of ProEGCG for Treatment of Endometriosis

Front Pharmacol. 2021 Oct 14:12:714790. doi: 10.3389/fphar.2021.714790. eCollection 2021.

Authors

Sze Wan Hung¹, Bo Liang^{1

2}, Yating Gao¹, Ruizhe Zhang^{1

3}, Zhouyurong Tan¹, Tao Zhang¹, Pui Wah Jacqueline Chung¹, Tak Hang Chan^{4

5}, Chi Chiu Wang^{1

6

7

8}

Affiliations

¹ Department of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China.
² Guangdong Provincial Key Laboratory of Marine Biotechnology, Institute of Marine Sciences, Shantou University, Shantou, China.
³ Center for Reproductive Medicine, Henan Key Laboratory of Reproduction and Genetics, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
⁴ Department of Applied Biology and Chemical Technology, State Key Laboratory of Chemical Biology and Drug Discovery, The Hong Kong Polytechnic University, Hong Kong, China.
⁵ Department of Chemistry, McGill University, Montreal, QC, Canada.
⁶ Reproduction and Development, Li Ka Shing Institute of Health Sciences, The Chinese University of Hong Kong, Hong Kong, China.
⁷ School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China.
⁸ Chinese University of Hong Kong-Sichuan University Joint Laboratory in Reproductive Medicine, The Chinese University of Hong Kong, Hong Kong, China.

Abstract

Endometriosis is defined as endometrial tissues found outside the uterine cavity. ProEGCG is a prodrug of Epigallocatechin gallate (EGCG), a potent polyphenol found in green tea. It inhibits the development of endometriotic lesions of mouse model in vivo, with higher efficacy and more remarkable anti-oxidative ability than EGCG. Our study aims to identify the molecular binding targets and pharmacological actions of ProEGCG in treating endometriosis. Protein target interaction study is essential to fully characterize the mechanism of actions, related therapeutic effects, and side effects. We employed a combined approach, starting with an in silico reverse screening of protein targets and molecular docking, followed by in vitro cellular thermal shift assay (CESTA) to assess the stability of protein-small molecule complexes. Then microarray and immunostaining of endometriotic lesions in mice in vivo confirmed the molecular interaction of the selected targets after treatment. Our study identified enzymes nicotinamide nucleotide adenylyltransferase (NMNAT)1 and NMNAT3 as protein targets of ProEGCG in silico and in vitro and were overexpressed after ProEGCG treatment in vivo. These findings suggested that participation in nicotinate and nicotinamide metabolism potentially regulated the redox status of endometriosis via its antioxidative capacities through binding to the potential therapeutic targets of ProEGCG.

Keywords: EGCG; NMNATs; antioxidants; endometriosis; prodrug; protein targets; reactive oxygen species; treatment.