GLI1 Inhibitors Identified by Target Protein Oriented Natural Products Isolation (TPO-NAPI) with Hedgehog Inhibition

This report describes the development of a target-protein-oriented natural-products-isolation (TPO-NAPI) method for Hedgehog inhibitors and the direct GLI1 inhibitor, 5′-O-methyl-3-hydroxyflemingin A (3), which inhibited hedgehog (Hh) signal transduction and diminished characteristics of cancer stem cells. Eight natural products (including three newly described products) that directly bind to GLI1 were rapidly obtained via the TPO-NAPI method developed using GLI1 protein-immobilized beads. 5′-O-Methyl-3-hydroxyflemingin A (3) inhibited Hh signaling (IC50 7.3 μM), leading to decreasing production of the Hh target proteins BCL2, PTCH1, and BMI1. 5′-O-Methyl-3-hydroxyflemingin A (3) was cytotoxic to Hh-related cancer cells. CD experiments revealed that 5′-O-methyl-3-hydroxyflemingin A (3) directly bound GLI1 (K d = 7.7 μM). Moreover, 5′-O-methyl-3-hydroxyflemingin A (3) diminished cancer stem cell characters of Huh7 such as sphere formation and production of the cancer stem cell marker EpCAM. These results suggest that Hh inhibitors can efficiently suppress the activity of cancer stem cells.