Discovery and evaluation of cytisine N-isoflavones as novel EGFR/HER2 dual inhibitors

Aberrant signaling of EGFR (ErbB) family members, in particular epidermal growth factor receptor (EGFR) and human epidermal growth factor 2 (HER2), is associated with the occurrence and development of many types of human malignancies (e.g., breast, lung, and gastric cancers), and dual targeting of EGFR/HER2 by small -molecular inhibitors has proven to be an effective therapeutic approach for treating these cancers. Herein we extracted and isolated from the medicinal plant Sophora alopecuroides L. a new natural product, dubbed Cytisine N-methylene-(4',7-dihydroxy-3'-methoxy)-isoflavone (CNI1) that features a unique molecular framework. Our biochemical kinase assay suggested that one of its derivative CNI3 exhibited the best, micromolar (mu M) inhi-bition activities against the EGFR (IC50 of 1.1 mu M; Ki of 0.6 mu M) and HER2 (IC50 of 3.5 mu M; Ki of 1.8 mu M) kinases. By contrast, another derivative CNI4 was most potent in inhibiting the EGFR-overexpressing A431 cancer cell line (IC50 of 45.5 mu M) and the HER2-overexpressing BT-474 cancer cell line (IC(50)of 32.9 mu M), while the respective cellular activities of Lapatinib (a marketed drug) were 24.9 and 20.3 mu M under the same assay condition. Moreover, both CNI3 and CNI4 showed desirable anti-metastatic efficacy in another two breast cancer models (viz., MDA-MB-231 and 4T1). In addition, we explored the inhibitory mechanisms of the CNIs against EGFR and HER2 by molecular dynamics simulation and revealed a novel mode of action that engages the cytisine and chromone moieties in CNIs. By combining structure-and ligand-based analysis, we further rationally engineered a new CNI compound that exhibits considerably improved cytotoxicity against both types of A431 and BT-474 cancer cells. Our study demonstrates the CNI compounds as a new class of EGFR/HER2 dual in-hibitors and paves a way for their further development.