Abstract

Medicinal herbs contain natural products (NPs) possessing rich scaffolds valuable for drug discovery, particularly in oncology. While most NP-derived cancer therapeutics directly kill tumor cells, emerging opportunities lie in modulating antitumor immunity. However, target-annotated NPs for cancer immunotherapy remain scarce. Herein we established a multiplexed platform combining virtual screening, affinity selection-mass spectrometry, and metabolomics profiling to identify bioactive NPs targeting the adenosine 2A receptor (A2AR), a master regulator of tumor immunosuppression. Screening the crude extract of a medicinal herb and isolating the active constituent resulted in the discovery of a novel dual antagonist for A2AR/A2BR with preferential activity on A2AR. This compound, ER-15, adopts a unique binding mode as revealed by structural modeling, MD simulations, mutagenesis, and SAR analysis. Functionally, ER-15 reversed adenosine-mediated immunosuppression and augmented the immune checkpoint inhibitor therapy in both the animal model and patient-derived tumor organoids, supporting its therapeutic potential in anti-PD-1-resistant tumors. Therefore, our strategy is expected to overcome traditional NP discovery bottlenecks, enabling efficient identification of target-annotated novel leads for drug development.