Screening of small molecule inhibitors against RhoA protein from Alisma using an online detection system

Targeted high-throughput screening of inhibitors from natural products is an effective approach in the treatment of cancer progression. RhoA protein is essential for many signaling pathways. It is closely related to the occurrence and development of tumor. So far, there are only a few reports on the screening of small molecule inhibitors of RhoA protein from natural products. In this study, an online UHPLC-PDA-MS²-RhoA-FLD screening system was established for the first time to identify RhoA inhibitors from medicinal Alisma. Using this online system by adding fluorescent probes protopine [LZ1] to proteins, 17 active components were identified from Alisma, including 14 terpenoids. Their binding abilities were evaluated by Surface Plasmon Resonance experiments. The activities of representative compounds were tested and showed anti-proliferative effect in cancer cells. Mechanistic studies showed that these compounds were able to downregulate the cellular expressions of RhoA associated proteins. This study provides potential lead compounds as small molecule inhibitors of RhoA protein for cancer therapy. This reported method can be used for targeted screening of small molecule inhibitors against tumors, and provides an approach for screening tumor inhibitors from natural products.