S9 Targets PI3K/mTOR/Tubulin Figure 1. S9 inhibits PI3K-Akt-mTOR signaling. A–B), S9 depresses EGF-triggered activation of PI3K-Akt-mTOR signaling pathway. Serum- deprived Rh30 cells (A) and SK-OV-3 cells (B) were treated with indicated concentrations of S9 for 1 h followed by EGF (50 ng/ml) stimulation for 10 min. Cells were harvested for Western blot analysis with antibodies specific for p-PDK1 (S241), PDK, p-Akt (T308), p-Akt (S473), Akt, p-mTOR (Ser2448), mTOR, p-p70S6K (T389), p70S6K, p-4E-BP1(T37/64), p-4E-BP1 (T70), 4E-BP1 and actin. Arrows indicate p70 isoform of S6 kinase protein. C) S9 blocks Akt membrane translocation and membrane ruffling. CHO (pCORON1000-EGFP-Akt) cells seeded on chamber were starved for 2 h then treated with 5 or 10 mM S9 for 1 h followed by IGF stimulation for 5 min. Fluorescent pictures were captured with confocal fluorescent microscopy. White arrows indicate cell membrane ruffling. Blue arrows indicate fluorescent foci. D) Total Akt granule intensity in each CHO (pCORON1000-EGFP- Akt) cell was counted with statistic module by IN Cell Analyzer 1000. Data shown are representative from two independent experiments. doi:10.1371/journal.pone.0004881.g001 binding site of p110, the catalytic subunit of PI3K [15], and that S9 is a pan-inhibitor for PIKK family members, molecular docking approach was performed to predict their potential binding sites and possible binding mode between S9 and p110a (Figure 2E). The docking results indicated that S9 could bind to the ATP pocket with high affinity. For a better resolution, a close-up view of the binding interface was developed. S9 forms one hydrogen-bond with the main-chain of Tyr 836 and hydrophobically interacts with Trp 780, Ile 800, Ile 848, Ile 850 and Val 851 of the enzyme. In addition, overall arrangements of S9 and ATP are very similar comparing their binding modes in the active site of PI3K. Likewise, the S9 hydrogen bonds to the Thr 2245, Lys 2187 and His 2447 of the enzyme in the mTOR-S9 complex (Figure 2F). Among them, Lys2187, which are highly conserved in PIKK PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4881