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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Nat Cell Biol. 2015 Oct 19;17(11):1484–1496. doi: 10.1038/ncb3255

Fig. 6.

Fig. 6

Identification of Physcion and its derivative S3 as 6PGD inhibitors (a) Upper: Screening strategy for lead compounds as 6PGD inhibitors. Lower: Structure of Physcion and its derivative S3. (b) Purified 6PGD (left) and G6PD (right) were assayed for 6PGD and G6PD activity, respectively, in the presence of Physcion. (c) Absolute IC50 values of Physcion and S3 were determined in activity assays using purified enzymes. (d) Kd values were determined for Physcion or S3 binding to purified human 6PGD proteins. The fluorescence intensity (Ex: 280nm, Em: 350nm) was measured 37. (e-f) Physcion-treated H1299 cells were assayed for 6PGD (e; left) and G6PD (e; right) activity, and cell viability (f). (g) Schematic representation of molecular docking study of Physcion based on the crystal structure of 6PGD (PDB code: 3FWN) in complex with its substrate 6-PG. Physcion (green) is docked in a pocket near the binding site of 6-PG (yellow) that is surrounded by residues including M15, K76, K261 and H452. (h) Purified 6PGD WT (left) and M15A mutant (right) were treated with Physcion and assayed for 6PGD activity. Absolute IC50 values are shown; NR=not reached. (i) H1299 6PGD knockdown cells were transfected with 6PGD WT (left) and M15A mutant (right), followed by cell proliferation assay based on cell numbers in the presence of Physcion. (j) Cell viability of Physcion-treated human cancer cells were determined by MTT assay. Normal proliferating human dermal fibroblasts (HDF) and melanocyte PIG1 cells were included as controls. (k) Cell proliferation rates of H1299 cells treated with Physcion were determined. (l) Apoptotic cell death (48 hours) of H1299 cells harboring AMPK shRNA or an empty vector in the presence of Physcion were determined by annexin V staining. Data are from a single experiment that is representative of 3 independent experiments for (b, e, h), 2 independent experiments for (f, i, l), and 4 independent experiments for (j-k). Source data for independent replications and experiments with sample size<5 are available in Supplementary Table 1. Uncropped Western blots are provided in Supplementary Figure 9.