Development and validation of bio-orthogonal probe to identify the molecular targets of (-)- epigallocatechin-3-gallate (EGCG), a major green tea polyphenol

Abstract

Dietary factors greatly influence the health of a human being. One of the most commonly used beverages is tea. It is the second-most frequently consumed drink worldwide next to the water. Tea plant, Camellia sinensis is being cultivated from thousands of years and its leaves are used for the medicinal purposes as they contain the healing ingredients. One of most health beneficial tea among all types is green tea. It is enriched with active polyphenols such as (-)-catechin, (-)- epigallocatechin (EGC), (-)-epicatechin-gallate (ECG), (-)- epigallocatechin-3-gallate (EGCG) and recent research works report that the green tea has therapeutic effect, majorly because of these polyphenols. Among all, EGCG is potentially chemotherapeutic and chemo preventive polyphenol. Currently EGCG is paid more attention on its anti-cancer property as it is acting as an anti-cancer agent to most prevalent cancers like breast cancer, gastric cancer, colorectal cancer, skin cancer, esophageal cancer, etc. But it is not being used as a drug for none of the cancers as the mode of action of EGCG is unknown. To unveil the mechanism of action, the molecular targets of EGCG in the cancer cells are to be known. Here, we are using a chemoproteomics platform to identify the interactome of EGCG. For this, we designed and synthesized an EGCG bio-orthogonal probe, namely, 4”-alkyne EGCG and characterized by LC-MS, HPLC and NMR. The probe, 4”-alkyne EGCG was validated and its properties were compared by acellular DPPH assay and cell proliferation assay by using crystal violet which was performed in MCF-7, HeLa and A549 cell lines. Then the copper (I)-mediated Click reaction was performed by treating the abovementioned cancer cell lines with 4”-alkyne EGCG and their protein lysates were used for the reaction with biotin azide. Then the SDSPAGE and Western blot were performed by using streptavidin and identified the proteins which were binding with 4”-alkyne EGCG and variations are observed in binding pattern of EGCG with proteins among the above-mentioned cell lines. The covalent binding of EGCG was also identified by NBT staining of EGCG & 4”-alkyne EGCG with cell lysates from cancer cell lines separately. Mass spectrometric analysis was performed to identify the covalent binding pattern of the EGCGglutathione adduct and 4”-alkyne EGCG-glutathione adduct. Visualization and identification of protein targets of EGCG in the cancer cell lines help in exploring the mechanism of action of EGCG. If the mode of action is known, the limitations of EGCG to be used as drug can be solved. The binding nature of EGCG with the proteins also play a major role in delivering its therapeutic properties. If the EGCG binds covalently it can permanently modify the proteins for their better function.