Fluorescence strategy for evaluating the effect of traditional Chinese medicine and its active ingredients on CD73 activity using supramolecular host-guest reporter pair

Traditional Chinese Medicine (TCM) contains numerous active ingredients with potential anti-cancer properties. Ecto-5’-nucleotidase (CD73) is an emerging cancer therapeutic target, catalyzing the production of extracellular adenosine within the tumor microenvironment, promoting immunosuppression. Consequently, the inhibition of CD73 is considered a promising strategy to block adenosine-mediated immunosuppression, emphasizing the urgent need to discover CD73 inhibitors within TCM. The objective of this study was to facilitate the screening and evaluation of TCM and its active ingredients. To this end, a straightforward, rapid CD73 inhibitor screening strategy was developed. This strategy was based on an indicator displacement assay (IDA) sensor. The construction of the optimized supramolecular reporter pair Azure B@tetralactam macrocycle (AB@TM), to achieve greater sensitivity, building on foundations established in our previous research. This enhanced sensitivity results from AB@TM exhibiting a substantially larger fluorescence signal change upon exposure to equivalent concentrations of guanosine (G) compared to the original sensor. Notably, this augmented response is discernible at considerably lower guest concentrations (as low as 0.5 μM). Using this enhanced system, the impact of 19 TCMs on CD73 activity was assessed, leading to the identification of
Curcuma longa
, Yanlu Rukang capsule, and Xuebijing injection (XBJ) as potent inhibitors. Subsequently, due to its relatively well-defined composition, XBJ was selected to investigate the inhibitory effects of its active ingredients on CD73. Among these components, Paeoniflorin demonstrated a potent, dose-dependent inhibitory effect, with an IC
50
of 0.2 ± 0.025 μM. The inhibitory effect of paeoniflorin was validated by a malachite green assay, and molecular docking simulations suggested potential interaction sites between paeoniflorin and the CD73 protein. In summary, this method establishes a reliable platform for screening CD73 inhibitors, aiding in the discovery of inhibitory TCMs and their active components, as well as the determination of IC
50
values. This approach benefits the development and modernization of TCM by providing efficient tools for bioactive compound identification.