Background: 2-Methoxystypandrone (2-MS), isolated from the roots of Polygonum cuspidatum, is a
potent dual inhibitor of the STAT3 and NF-κB pathways.
Objective: To investigate the molecular targets and mechanisms of 2-MS.
Method: A biotin-conjugated 2-MS analog, named 2-MS-Biotin, was designed and synthesized. The effects of
2-MS-Biotin on the STAT3 and NF-κB pathways were examined by Western blotting. The cytotoxicity of 2-
MS-Biotin was evaluated using real-time cell analysis system. Proteins directly bound to 2-MS-Biotin were
pulled down through streptavidin agarose beads and were detected using Western blotting.
Results: 2-MS-Biotin retained the inhibition activities of the parent compound 2-MS on the STAT3 and NF-κB
pathways as well as on cancer cell growth. Also, JAK2 and IKK proteins can be effectively pulled down by 2-
Conclusion: Using 2-MS-Biotin as a tool, both JAK2 and IKK were identified as the targets of 2-MS.