Methods Fluorescence polarisation assay The Bid BH3 domain peptide was synthesised and labelled inhibitor Ganetespib with 5 Carboxyfluorescein at the N terminus. For the competitive binding assay, 200 nM Bcl xL, Bcl 2, or Mcl 1 was mixed with various concentrations of com pounds in PBS. After incubation for 1 h at 37 C, an equal volume of 200 nM 5 FAM labelled BH3 peptide was added to the solution. After incubation for 10 min at 37 C, the fluorescence polarisa tion was measured on a TECAN Genios Pro microplate reader. The excitation wavelength and emission wave length were set to 485 nm and 535 nm, respectively. The 50% inhibiting concentration value was analysed by the GraphPad Prism program. The Ki was calculated by a web based tool. Molecular modelling The refined structure of Bcl xL was used for prediction binding mode between Jac A with Bcl xL.

The program Maestro 9. 0 was used for this assessment. All water molecules were removed from the structure of the complex. Hydrogen atoms and charges were added during a brief relaxation that was performed using the Protein Preparation Wizard workflow in Maestro 9. 0. After optimising the hydrogen bond network, the crystal structure was minimised using the OPLS 2005 force field with the maximum root mean square deviation value of 0. 3. The grid enclosing box was centred on the ligand ABT 737 in the refined crystal structure as described above, and defined so as to enclose the resi dues located within 14 from the ligand. This domain has been identified as the BH3 domain, which is the fun damental motif for dimerization with the BH3 peptide.

The three dimensional structure of Jac A was generated with the Ligprep module. Docking process was per formed using GLIDE with default docking parameter setting with extra precision approach. Cell culture Cell lines MBA MB 231, T47D, LOVO, A549, HepG2, K562, HL 60, and THP 1 cells were obtained from the American Type Culture Collection. All cell culture supplies were obtained from Invitrogen. Thiazolyl blue tetrazolium bromide and dimethyl sulfoxide were purchased from Sigma Aldrich. Cells were cultured in RPMI 1640, IMDM, or DMEM and maintained in a Thermo incu bator with humidified air containing 5% CO2 at 37 C. All culture media contained 10% FBS and 1% penicillin streptomycin. Cytotoxicity assay The cytotoxic activitiy of Jac A against human cancer cells was measured by the MTT colorimetric assay.

Four thousand cells were seeded in 96 well plates and treated GSK-3 with the compounds for 48 h at serial con centrations. Then, 10 uL MTT solution was added to each well, and the plates were incu bated for an additional 2 4 h at 37 C. The supernatant was carefully removed, and 100 uL DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was recorded on a BioTek Synergy 2 plate reader.