Naturally Occurring Homoisoflavonoids Function as Potent Protein Tyrosine Kinase Inhibitors by c-Src-Based High-Throughput Screening

Protein tyrosine kinase (PTK) inhibitors represent emerging therapeutics for cancer chemoprevention. In our study, hematoxylin (<b>26</b>) was identified as one of the most remarkable c-Src inhibitors in an orthogonal compound-mixing library (32200 compounds) by using an ELISA-based automated high-throughput screening (HTS) strategy. Interestingly, hematoxylin was found to be an ATP competitive broad-spectrum PTK inhibitor in vitro, with IC<sub>50</sub> values ranging from nanomolar to micromolar level. Further studies showed that such inhibition was associated with the PTK phosphorylation and subsequent downstream signaling pathways. The structure−activity relationship assessment of the PTK inhibitory potency of hematoxylin analogues isolated from <i>Heamatoxylon campechianum</i> was in good agreement with the result of concurrent molecular docking simulation: the catechol moiety in ring A and the hematoxylin-like three-dimensional structure were essential for c-Src-targeted activities. Hematoxylin and its natural analogues were substantially validated to function as a new class of PTK inhibitors.