The Synergy of Probe-Competitive Chip and Automated Platform in Defining Screening Scope and Uncovering Target Inhibitors

Herbal medicines contain complex bioactive components, yet robust methods for directly screening compounds from herbal extracts that target specific biological targets are still lacking. This study aimed to establish a method for screening active compounds from herbal extracts. A competitive solid-phase kinase target chip was developed using an alkynyl-modified XO44 probe. Using the kinase MAPK1 as an example, the MAPK1 target chip was employed to screen 15 herbal extracts. After identifying a candidate herbal extract, semi-preparative chromatography coupled with protein thermal stability analysis was used to separate compounds from the selected extract. An automated chemobiology platform was then applied for high-throughput screening of the isolated components to identify MAPK1 inhibitors. Subsequently, CETSA, BLI, and molecular docking were utilized to validate the binding of the screened active compound. The anti-lung cancer effects of the compound were further evaluated at the cellular level, in patient-derived lung cancer organoids, and in subcutaneous xenograft mouse models. Using the competitive kinase chip, Inula helenium L. (elecampane) was identified from the 15 herbal extracts as capable of competing with the XO44 probe. After optimizing and standardizing the automated platform (> 1,000 reactions/hour), high-throughput screening of the semi-preparatively separated components from Inula helenium L. identified alantolactone as a novel MAPK1 inhibitor. Further validation confirmed the direct binding of alantolactone to MAPK1 and STAT3, and demonstrated its ability to sensitize A549/CDDP cells, enhance toxicity in patient-derived lung cancer organoids, and improve pharmacological efficacy in vivo . By integrating protein chips with a robotic automated chemobiology platform (ChiRob), these results establish ChiRob as an efficient and implementable platform for the target-directed discovery of bioactive compounds from complex herbal mixtures. • Development of a click chemistry-enabled kinase chip designed for competitive screening, which overcomes limitations associated with traditional affinity ultrafiltration methods. • Construct a high-throughput screening (HTS) strategy that combines protein chips with a robotic automated chemobiology platform (ChiRob) to screen active ingredients in complex systems such as herbs. • Discovery of alantolactone (from Inula helenium L.) as a novel MAPK1/STAT3 inhibitor, with validation in cisplatin-resistant A549 cells and patient-derived lung organoids. • New in vivo data demonstrating the platform’s utility in identifying physiologically relevant inhibitors.