SUPPLEMENTARY INFORMATION. Small molecule activation of the TRAIL receptor DR5 in human cancer cells

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1 SUPPLEMENTARY INFORMATION Small molecule activation of the TRAIL receptor DR5 in human cancer cells Gelin Wang 1*, Xiaoming Wang 2, Hong Yu 1, Shuguang Wei 1, Noelle Williams 1, Daniel L. Holmes 1, Randal Halfmann 1, Jacinth Naidoo 1, Lai Wang 4, Lin Li 4, She Chen 3, Patrick Harran 5, Xiaoguang Lei 2,3*, and Xiaodong Wang 1,3* 1 Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA. 2 School of Pharmaceutical Science and Technology, Tianjin University, Tianjin , China 3 National Institute of Biological Sciences (NIBS), Beijing , China 4 Joyant Pharmaceuticals, Dallas, TX 75207, USA 5 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA (*) Corresponding author: wangxiaodong@nibs.ac.cn, leixiaoguang@nibs.ac.cn, and gelin.wang@utsouthwestern.edu, Tel: , Fax:

2 SUPPLEMENTARY RESULTS Supplementary Figure 1. Schematic of the screen design and procedure. 2

3 Supplementary Figure 2. Bioymifi efficiently kills a variety of cancer cells as singleagent. The dose-response curves for bioymifi or TR in the absence or presence of SM were plotted for HeLa, H460, U2OS, HT29, H1155, and Miapaca cell lines. Supplementary Figure 3. Full-length gel images of western blot depicted in the Figure 2 of main text. Supplementary Figure 4. DR5 sirnas specifically decrease the mrna levels of DR5 but not DR4. The DR5 and DR4 mrna levels in the knockdown cells were analyzed using quantitative RT-PCR. 3

4 Supplementary Figure 5. Sensitivity to bioymifi is similar in HCC15 parental cells and the DR5-overexpressing cells (15-13). The cells were treated with the indicated concentrations of bioymifi in the presence of SM for 48 hours. Supplementary Figure 6. Full-length gel images of western blot depicted in the Figure 3 of main text. 4

5 Supplementary Figure 7. (a) Both FADD and TRADD are required for TRAIL-induced apoptosis in MEFs. MEFs derived from WT, FADD -/-, or TRADD -/- mice were treated with 100ng/ml mouse TRAIL for 48 hours. The cell viability was measured by using the Cell Titer-Glo kit. (b-c) T98G cells were transfected with Luciferase and RIPK1 sirnas. After 48 hours, cell viability was measured, and the cell lysates were subjected to the western blot with RIPK1 and tubulin antibodies. Supplementary Figure 8. TRAIL mrna levels in the knockdown cells. T98G were transfected with three independent sirnas (TR-1, -2 or -3) for TRAIL. After 48 hours of transfection, RT-PCR was performed to assess the knockdown efficiency. 5

6 Supplementary Figure 9. A Coomassie-stained SDS-polyacrylamide (PAGE) gel showing purified recombinant proteins for His-tagged extracellular domain of DR4 (DR4-ECD) and DR5 (DR5-ECD). Supplementary Figure 10. A dilution series for bioymifi was produced from a 10mM DMSO stock into TBS buffer. After 1 hour incubation at room temperature, the solutions were centrifuged at 12,000rpm for 5 min. The absorbance for the supernatant of these samples was measured at 420 nm, which is the peak absorbance wavelength in TBS solution. 6

7 Supplementary Figure 11. Full-length gel images of western blot depicted in the Figure 5 and Figure 6 of main text. Supplementary Figure 12. Bioymifi stimulates high molecular weight complex formation. DR5-expressing cells were stimulated or not for 2 hours with 10 µμ bioymifi 7

8 in the absence or presence of SM. The cell lysates were fractionated on a Superdex-200 gel filtration column. Flag-DR5, FADD, and Caspase-8 were analyzed by western blotting. The size of the complex can be estimated by the molecular weight of the standard markers for size exclusion column indicated at the top of the figure. Supplementary Figure 13. Depletion of DR5 reduces the receptor clustering induced by bioymifi. (a) The DR5-expressing cells transfected with control or DR5 sirnas were treated with 10 µm bioymifi for 1 hour and then immunostained with a Flag antibody (red). The images were acquired using a DeltaVision microscope. The exposure time was 0.5 sec, and the images were processed using Image J software. Scale bar: 20 µm. (b) Cell lysates of Luciferase RNAi and DR5 RNAi cells were analyzed by western blot with Flag and actin antibodies. 8

9 Supplementary Figure 14. Bioymifi-induces aggregation is specific for DR5. (a) DR5-Flag-expressing cells were incubated for 1 hour with DMSO (control) or 5 µm bioymifi. The cells were immunostained with Flag, DR4, FAS, or TNFR1 antibodies. The images were taken using a Zeiss LSM 510 confocal microscope. Scale bar represents 10 µm. The percentage of the cells with clustering death receptor were quantified in (b) (n= 100). 9

10 Supplementary Table 1. SAR analysis identified an active compound, bioymifi, with a well-defined structure. Twenty-four chemical analogs of A2C2 were compared based on cell-killing efficacy. The dose-response curve was generated for each compound based on cell survival as measured using the Cell Titer-Glo Luminescent Cell Viability Assay. IC 50 values represent the concentration of compound that was required for 50% inhibition of cell viability. IC 50 values of cell viability were determined in T98G cells. 10

11 Supplementary Table 2. Kd (binding constant) and IC 50 values of the compounds used in the Figure 5c of main text. 11