5 106 K562 cells were subcutaneously injected in the right flank of mice. When the tumours reached approximately 200 mm3, the mice were randomly divided into four groups and treated with Jac A at 2, 10, 50 mg/kg or vehicle by oral gavage. Tumour growth was monitored by measuring the tumour size twice a week for 3 weeks after treatment. A nothing digital calliper was used to measure the tumour in two or thogonal dimensions. The volume was calculated with the formula 2/2. The body weight and survival of the nude mice were moni tored throughout the experiments. All animal experiments were approved by the animal care committee of the Sec ond Military Medical University in accordance with insti tutional and Chinese government guidelines for animal experiments.

Statistical analysis The data from the in vitro and in vivo experiments at dif ferent time points for the different treatment groups were analysed for statistical significance with the GraphPad Prism program. One way ANOVA was used among groups, followed by the Mann Whitney U test for post hoc comparisons to determine the P values. The statistical significance of differences in the survival of mice from the different groups was deter mined by the log rank test using the same program. Chemistry The purity of Jac A was verified with NMR and HPLC, and the purity of Jac A was 97%. Jacarelhyperol A, isolated from Hypericum japonicum Thunb. ex Murray. yellow powder Results Screening active compounds Fuoresence polarization was used to screen for Bcl 2 protein inhibitors. Jac A was chosen as the candidate compound for further research because of its high affinity to Bcl 2 proteins.

As shown in Figure 1 and Table 1, Jac A can dose dependently bind to Bcl xL, Bcl 2, and Mcl 1 with a Ki value of 0. 46 uM, 0. 43 uM, and 1. 69 uM, respect ively, which near to the activity of positive control Gossypol, a known Bcl 2 protein inhibitor. Predicting the binding modes of Jac A with Bcl xL To map the binding site of Jac A, we built complex struc ture of the compound with Bcl xL by docking. Jac A contains two xanthones and can generates many conformations by rotating the C10 ? O and O ? C3�� bonds. The best binding model is shown in Figure 2A, in which the two xanthones of Jac A exhibit two different orientations with a 90 dihedral angle and occupy three sub pockets. The three sub pockets play an important role in binding with pro death BH3 only proteins and ligands.

The P2 pocket, formed by residues Tyr 101, Ala 104, Leu 108, Val 126, Asn 135, Ala 142, and Ser 145, makes hydrophobic contacts with Carfilzomib the aro matic H ring of Jac A. In the P4 pocket, the aromatic B ring, double bond of the A ring, and the two methyl groups at C 3 make hydrophobic contacts with the hydro phobic pocket formed by residues Glu 96, Phe 97, Gly 138, and Tyr 195.