Anticancer osmium complex inhibitors of the HIF-1 and p300 protein-protein interactionChao Yang1,*, Wanhe Wang2,*, Guo-Dong Li1, Hai-Jing Zhong1, Zhen-Zhen Dong2, Chun-Yuen Wong3, Daniel W. J. Kwong2, Dik-Lung Ma2 & Chung-Hang Leung11State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China2Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China3Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR, China*These authors contributed equally to this work.Correspondence and requests for materials should be addressed to C.-H.L. (email: duncanleungumac.mo) or D.-L.M. (email: edmondmahkbu.edu.hk)SUPPLEMENTARY FIGURES Supplementary Figure 1. HIF-1 expression under normoxic and hypoxic conditions. Normoxic and hypoxic MC3T3-E1 cell extracts were prepared and protein samples were collected. Western blotting analysis was performed.Supplementary Figure 2. HIF-1 expression under normoxic and hypoxic conditions. Normoxic and hypoxic HEK293T cell extracts were prepared and protein samples were collected. Western blotting analysis was performed.Supplementary Figure 3. Dose-dependent effect of chemotin, complexes 27 and phen-tMe ligand on HRE activity as determined by a dual luciferase reporter assay. Hypoxic MC3T3-E1 cells were treated with indicated concentrations of chemotin (a), complexes 27 (b-g) and ligand (c) for 16 h. IC50 value of chemotin is ca. 9.83 M, IC50 values of complexes 27 and phen-tMe ligand were above 10 M. Error bars represent the standard deviations of results obtained from three independent experiments.Supplementary Figure 4. 1H NMR spectra of complex 1 at a concentration of 5 mM in 90% d6DMSO/10% D2O at 298 K over 7 days.Supplementary Figure 5. UV/Vis absorption of complex 1 at a concentration of 20 M in 80% acetonitrile/20% Tris-HCl buffer (20 mM containing 20 mM NaCl, pH 7.5) at 298 K over 7 days.Supplementary Figure 6. Low-energy binding conformations of (a) p300 and 1, (b) HIF-1 and p300 and (c) superimposition of (a) and (b) as generated by molecular docking. The protein is depicted as a ribbon representation (red and purple), while 1 is depicted as a space-filling representation showing carbon (beige) and nitrogen (blue).Supplementary Figure 7. Densitometry analysis of the cell-based Western blot showing complex 1-mediated reduction of HIF-1-p300. Approximate IC50 value = 0.41 M.Supplementary Figure 8. The cytotoxicity effect of neat water or 0.1% DMSO in water on MC3T3-E1 cells under (a) normoxic and (b) hypoxic conditions as determined by a MTT assay. MC3T3-E1 cells were incubated with neat water or 0.1% DSMO in water for 48 h under normoxic or hypoxic conditions. Control is the fresh DMEM medium with 1% penicillin containing 1% FBS.Supplementary Figure 9. The cytotoxicity effect of complex 1 on (a) A498 cells, (b) HepG2 cells and (c) MC3T3-E1 cells under normoxic and hypoxic conditions as determined by an MTT assay. Cells were exposed to the indicated concentrations of complex 1 for 48 h. Complex 1 inhibited the growth under normoxic and hypoxia conditions of A498 cells with IC50 values of 36.8 M and 31.1 M, respectively, HepG2 cells with IC50 values of 49.6 M and 36.2 M, respectively, and MC3T3-E1 cells with IC50 values of 10.2 M and 4.2 M, respectively. Error bars represent the standard deviations of the results from three independent experiments.Supplementary Figure 10. The intracellular ROS production were determined by flow cytometry analysis. MC3T3-E1 cells were treated with 0.1% DMSO or 3 M of complexes for 16 h under hypoxia conditions. Cells were analyzed by flow cytometry upon DCFH-DA staining. (a) Flow cytometric histogram. (b) Quantification of mean fluoresence intensity.Supplementary Figure 11. Effect of complex 1 on NF-B activity as determined by a dual luciferase reporter assay. Hypoxic MC3T3-E1 cells were treated with indicated concentrations of complex 1 or chemotin (10 M) stimulation with TNF- (25 ng/mL) for 16 h. Error bars represent the standard deviations of results obtained from three independent experiments.abcdSupplementary Figure 12. HPLC trace for complexes 2-5. (a) complex 2 (1040% acetonitrile with 0.1% TFA over 10 min, 4065% acetonitrile with 0.1% TFA from 10 min to 15 min, and 4065% acetonitrile with 0.1% TFA from 15 min to 25 min); (b) complex 3 (1040% acetonitrile with 0.1% TFA over 10 min, 4065% acetonitrile with 0.1% TFA from 10 min to 15 min, and 4065% acetonitrile with 0.1% TFA from 15 min to 25 min); (c) complex 4 (1030% acetonitrile over 5 min, 3090% acetonitrile from 5 min to 15 min, and 9095% acetonitrile from 15 min to 25 min); (d) complex 5 (1030% acetonitrile over 5 min, 3090% acetonitrile from 5 min to 15 min, and 9095% acetonitrile from 15 min to 25 min).