Design, Synthesis And Characterization Of Small-Molecule Probes Of STAT3Signaling Pathway Inhibitor KT45004Isolated From Polygonum Cuspidatum

Objective:The present study aims at optimization of the isolation conditions of the STAT3signaling pathway inhibitor KT45004from the extracts of the roots of Polygonum cuspidatum, and then we attempt to design and synthesize the biotinylated, photoaffinitiy-labelled and fluorescent chemical probes in order to identify the cellular target proteins and the reactive residues of STAT3signaling pathway inhibitor KT45004.Methods:①Various column chromatographic techniques, such as silical gel, sephedex LH-20and preparative HPLC, were performed to isolate the STAT3signaling inhibitor KT45004from the extracts of the roots of Polygonum cuspidatum;②Based on the preliminary structure-activity relationship studies, a series of the biotinylated, photoaffinitiy-labelled and fluorescent chemical probes were designed and synthesized to identify the direct binding targets of STAT3signaling inhibitor KT45004;③Their chemical structures were characterized by NMR and MS spectroscopy;④Confocal laser scanning microscopy was applied to investigate the intracellular distribution and localization patterns of fluorescent probe SM40801in huamn liver cancer cells and cancer stem cells;⑤The cytotoxic activities of chemical probes against human liver cancer Huh-7cells were conducted using MTT assay.Result:①After optimization of the isolation conditions,463.4mg of bioactive compound KT45004(>98%purity) was prepared from the extracts of the roots of Polygonum cuspidatum, the yield of KT45004was0.24%;②Biotin-tagged affinity probe SM42504was synthesized with KT45004as the bioactive group, biotin as the reporter group;③Fluorescent probe SM40801was synthesized with KT45004as the bioactive group, egonol as the reporter group. Confocal laser scanning microscopy images showed the cytoplasmic and nucleus localization patterns of the cell-penetrating fluorescent probe SM40801, which displayed green fluorescence in human liver cancer cells and cancer stem cells;④Biotin-tagged affinity probe SM42504and fluorescent probe SM40801did not show cytotoxic activities towards human tumor Huh-7cells in the test range up to30μM.Conclusion:In the present study, we obtained the optimized conditions for isolation of STAT3signaling pathway inhibitor KT45004from the EtOAc extracts of the roots of Polygonum cuspidate; The biotinylated and fluorescent chemical probes were designed and synthesized to identify their target proteins; The fluorescent probe SM40801distributed throughout the cytoplasm and nucleus and displayed green fluorescence in human liver cancer cells and cancer stem cells; These chemical probes offered great advantages in target identification of KT45004, and this investigation may provide helpful guidance to target protein identification and molecular mechanisms investigation of bioactive natural products.