Synthesis of Biotinylated 2-methoxystypandrone and Identification of JAK2 and IKK as its Targets.

Background: 2-Methoxystypandrone (2-MS), isolated from the roots of Polygonum cuspidatum, is a potent dual inhibitor of the STAT3 and NF-kappa B 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-kappa B pathways were examined by Western blotting. The cytotoxicity of 2MS-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-kappa B pathways as well as on cancer cell growth. Also, JAK2 and IKK proteins can be effectively pulled down by 2MS-Biotin. Conclusion: Using 2-MS-Biotin as a tool, both JAK2 and IKK were identified as the targets of 2-MS.