Four Novel ROCK1 Inhibitors Suppressing Cytoskeleton and Movement in Breast Cancer Cells

Rho‐associated coiled‐coil protein kinase 1 (ROCK1), a key downstream effector of the Rho GTP‐binding protein within the Ras superfamily, regulates cellular metabolism, growth, differentiation, and signaling pathways associated with various disease. We identified four novel ROCK1 inhibitors through virtual screening technology and enzymatic activity assays—bilobetin, SCH 772984, puerarin 6''‐O‐xyloside, and GSK 650394. Their IC50 values were 11.82 μM, 12.19 μM, 15.27 μM, and 18.09 μM, respectively. To evaluate their ROCK1 related efficacy, we assessed their effects on the proliferation, cytoskeletal organization, migration, and invasion of MDA‐MB‐231 breast cancer cells. These compounds effectively reduced cell viability with IC50 values ranging from 20 μM to 32 μM. Additionally, a marked decrease in EdU uptake confirmed their potent inhibition of cell proliferation. Confocal fluorescence imaging revealed that suppression stems primarily from cytoskeletal disruption, thereby impairing migration and invasion, with in vitro inhibition rates of 70–85% and 69–86%, respectively. These findings not only enrich the types of ROCK1 inhibitors, but also provide novel molecular scaffolds for the development of anti‐breast cancer drugs.