Target identification studies of the antiproliferative natural product OSW-1

OSW-1 is a plant-derived natural product with strong antiproliferative activity in cancer cell lines and an unknown mechanism of action. OSW-1 was tested in the NCI's In Vitro Cell Line Screening Project and produced a pattern of cytotoxicity that statistically correlated with members of the cephalostatin, ritterazine, stellettin and schweinfurthin natural product families. These compounds we call cephalostatin 1-related antiproliferative molecules (CRAMs) do not show statistical correlation with other compounds and therefore are likely to have a unique mechanism of action. Our goal in this project was to identify the protein target of OSW-1 and provide evidence that the CRAMs share a unique mechanism of action. We found that all tested CRAMs show substantial differential cytotoxicity in an isogenic cell line differing in p21 expression, an effect not seen with other compounds. This indicated that they operated by a shared and unique mechanism of action. We then focused on identifying the protein target of OSW-1. We prepared an analog of OSW-1 that retained the potency of the parent compound but could be attached to a solid support to create an affinity matrix. Affinity chromatography identified oxysterol binding protein (OSBP) as a specific binder of OSW-1. Two experiments were performed to verify OSBP as the protein target of the CRAMs. One experiment showed a shRNA knockdown of OSBP in HCT-116 p21-/- and HeLa cell lines led to a 5.7 and 4.7 fold decrease in the GI50 of OSW-1, respectively. The GI50 of CRAMs ritterazine B and schweinfurthin A in HCT-116 p21-/- were reduced 8.3 and 9.2 fold, respectively, as well. A second experiment used immunofluorescence to show CRAMs change OSBP's subcellular localization from a cytosolic distribution to the Golgi membrane. These experiments support the hypothesis that the CRAMs share a mechanism of action that is dependent on OSBP.