Kusunokinin, a lignan compound, inhibits cancer cell proliferation and induces 
apoptosis; however, the role of kusunokinin is not fully understood. Here, we 
aimed to identify a target protein of (-)-kusunokinin and determine the protein 
levels of its downstream molecules. We found that (-)-kusunokinin bound 5 
possible target proteins, including CSF1R, MMP-12, HSP90-α, CyclinB1 and MEK1 
with ΔGbind less than -10.40 kcal/mol. MD simulation indicated (-)-kusunokinin 
and pexidartinib (P31, a specific CSF1R binding compound) shared some extents of 
functional similarity in which (-)-kusunokinin bound CSF1R at the juxtamembrane 
(JM) region with aromatic amino acids similar to pexidartinib using π-π 
interaction, as well as hydrogen bond. Both P31 and (-)-kusunokinin moved into 
the same CSF1R region and W7 was a mutual key residue. However, the P31 binding 
site differed from the (-)-kusunokinin binding site. For in vitro study, the 
synthetic (±)-kusunokinin exhibited stronger cytotoxicity than 
picropodophyllotoxin, silibinin and etoposide on MCF-7 cells and represented 
less toxicity than picropodophyllotoxin and doxorubicin on L-929 and MCF-12A 
cells. Knocking down CSF1R using a specific siRNA combination with 
(±)-kusunokinin demonstrated levels of cell proliferation proteins slightly 
higher than siRNA-CSF1R treatment. However, siRNA-CSF1R combination with P31 
represented the number of cell viability and cell proliferation proteins, like 
in the control groups (Lipofectamine and siRNA-Luciferase). Moreover, 
(±)-kusunokinin suppressed CSF1R and its downstream proteins, including AKT, 
CyclinD1 and CDK1. Meanwhile, both P31 and siRNA-CSF1R dramatically suppressed 
CSF1R, MEK1, AKT, ERK, CyclinB1, CyclinD1 and CDK1. Our overall results indicate 
that the mechanism of (±)-kusunokinin differed fairly from P31. We have 
concluded that (±)-kusunokinin inhibited breast cancer cell proliferation 
partially through the binding and suppression of CSF1R, which consequently 
affected AKT and its downstream molecules.