FIGURE 4 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Interaction of BFC1108 with Bcl-2. A, Limited proteolysis of Bcl-2 loop domain in the presence of BFC1108. Purified GST-tagged Bcl-2 loop domain and GST only control were incubated with 50 µmol/L BFC1108. The proteolysis pattern of loop domain was determined at the indicated times upon coincubation with trypsin. B, BFC1108 stabilizes and increases melting temperature (Tm) of Bcl-2 full-length protein. Thermal unfolding of Bcl-2 full-length protein in the presence of BFC1108 was monitored by SYPRO Orange fluorescence. Z-tag and no protein control samples were included to confirm BFC1108 specific interaction with Bcl-2. Two-way ANOVA with Sidak multiple comparisons post hoc test, **, P P C, BFC1108 stabilizes and increases Bcl-2 loop domain melting temperature (Tm). Thermal unfolding of Bcl-2 loop domain in the presence of BFC1108 monitored by SYPRO Orange fluorescence. One-way ANOVA with Dunnett multiple comparisons post hoc test, ****, P D, MDA-MB-231/Bcl-2, H460 cells were exposed to BFC1108 at 10 µmol/L concentration for 48 hours in a medium containing 10% serum. Change in conformation of Bcl-2 was determined by using Bcl-2 BH3 antibody followed by flow cytometric analysis.</p

Supplemental Figure 2 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Bioluminescent imaging of data in Figure 7 for individual mouse treated with vehicle or BFC1108 is shown.</p

FIGURE 1 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Bcl-2–dependent effects of BFC1108. A, Structure of BFC1108 (5-chloro-N-(2-ethoxyphenyl)-2-[(4-methoxybenzyol)amino]benzamide). B, Left: Bcl-2 expression in MDA-MB-231 cells transfected with pcDNA control vector (MDA-MB-231/Vector) or Bcl-2 expression vector (MDA-MB-231/Bcl-2) was determined by immunoblotting. Right: Cells were exposed to BFC1108 in a medium containing 10% FBS for 48 hours and viability was determined. ***, P C, Left: Bcl-2 expression in MCF-7 cells transfected with pcDNA control vector (MCF-7/Vector) or Bcl-2 expression vector (MCF-7/Bcl-2) was determined by immunoblotting. Right: Cells were exposed to BFC1108 in a medium containing 10% FBS for 48 hours and viability was determined. **, P D, Left: Bcl-2 expression in Jurkat cells transfected with control vector (Jurkat/Vector) or Bcl-2 expression vector (Jurkat/Bcl-2) was determined by immunoblotting. Right: Cells were exposed to BFC1108 in a medium containing 10% FBS for 48 hours and viability was determined. ***, P E, Left: Knockdown of Bcl-2 in MDA-MB-468 cells was determined by immunoblotting. Right: Bcl-2 siRNA and control siLuc siRNA transfected MDA-MB-468 cells were treated with BFC1108 in a medium containing 10% FBS for 48 hours. *, P < 0.05.</p

FIGURE 7 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Inhibition of breast cancer lung metastasis by BFC1108. A, BFC1108 suppresses the growth of lung metastasis in vivo. LMD-231 cells (200,000) stably expressing luciferase were injected into the tail vein of 6-week-old nude mice. Lung metastasis was detected in 2 weeks. Mice were randomized and were treated four times a week with vehicle or 100 mg/kg BFC1108 by intraperitoneal route (n = 8 for vehicle treatment and n = 9 for BFC1108 treatment). Bioluminescent imaging was performed once a week and quantified. *, P P P Supplementary Fig. S2. B, Representative mouse image from vehicle and BFC treatment is shown. C, Treatment with BFC1108 does not alter body weight of mice. Body weights were measured each time the mice were imaged. D, Suppression of lung metastasis by BFC1108. H&E staining showed increased number of tumor cells in lung tissue of mice with vehicle treatment, compared with BFC1108 treatment, at the end of the study. E, BFC1108 reduces proliferation of lung metastatic TNBC cells. Detection of Ki-67 indicating proliferating tumor cells in lung tissue from vehicle and BFC1108-treated mice.</p

FIGURE 3 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

BFC1108 activates intrinsic mitochondrial death pathway. A, BFC1108 suppresses the colony-forming ability of MEF cells in a Bcl-2–dependent manner. WT and Bcl-2−/− MEF cells were treated for 48 hours in a medium containing 10% FBS and the colony formation was determined after 2 weeks. B, Quantification of colony formation data shown in A. **, P C, Bax or Bak is required for BFC1108-induced suppression of viability. WT MEF, Bax Knockout (Bax−/− Bak+/+), Bak knockout (Bax+/+ Bak−/−) and double knockout (Bax−/− Bak−/−) MEF cells were treated with 1 µmol/L BFC1108 for 24 hours in 10% FBS medium and viability was assessed using CellTiter-Glo assay. **, P P D, BFC1108 decreases mitochondrial membrane potential of Bcl-2–expressing H460 lung cancer cells. JC-1 dye was used to stain live H460 cells that were treated with 10 µmol/L BFC1108 for 16 hours in 10% FBS containing medium. Images taken with FITC, and rhodamine filters were overlaid. Cells stained orange have intact mitochondrial outer membrane and the ones turning green have compromised outer membrane indicating loss of membrane potential.</p

FIGURE 5 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

BFC1108 induces cell death across multiple Bcl-2–expressing TNBC cell lines to a greater extent than ABT199. Cell death percentage is calculated based upon viability assay after 72 hours treatment at the indicated BFC1108 concentrations.</p

FIGURE 2 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

BFC1108 suppresses viability of multiple cancer cell types. A, Bcl-2–selective inhibition of clonogenic survival of MDA-MB-231 cells. MDA-MB-231/Vector and MDA-MB-231/Bcl-2 cells were exposed to 10 µmol/L concentration in medium containing 10% FBS for 48 hours and the colony formation was determined after 2 weeks. B, Quantification of colony formation data shown in A. ***, P C, Bcl-2–dependent induction of apoptosis by BFC1108. MDA-MB-231 cells with high or low Bcl-2 expression were treated with 10 µmol/L BFC1108 in a medium containing 10% FBS for 48 hours and apoptosis was determined by annexin V staining. Data from one representative experiment is shown. Refer to Supplementary Fig. S1 for additional independent experiments. D, BFC1108 inhibits viability of breast cancer cells with Bcl-2 expression. The indicated breast cancer cells were treated for 48 hours, and viability was measured using CellTiter-Glo assay. Data were analyzed by two-way ANOVA and Dunnett multiple comparison post hoc test, **, P P 50). E, BFC1108 suppresses viability of multiple cancer cell types with minimal effects on MCF10A nontransformed breast epithelial cells. Cells were treated for 48 hours in 10% serum containing medium and cell viability was determined. ***, P < 0.001.</p

FIGURE 6 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Antitumor effects of BFC1108 in an orthotopic breast cancer model. A, BFC1108 inhibits growth of high Bcl-2–expressing MDA-MB-231 TNBC cells in vivo. A total of 106 MDA-MB-231/Bcl-2 cells were implanted in the mammary fat pad of NOD.SCID mice (n = 8 per group). Once palpable tumors were detected, mice were randomized and treated with vehicle or BFC1108 at 100 mg/kg twice a week by intraperitoneal route. Tumor measurements were made with digital calipers twice a week. *, P B, The body weights of mice treated with vehicle or BFC1108 in A were measured twice a week. C, Induction of apoptosis of tumor cells by BFC1108 in vivo. Immunofluorescence was performed on frozen tumor sections to determine apoptosis by TUNEL staining. D, BFC1108 induces conformation change of Bcl-2 in tumor tissues. Bcl-2 conformational change was detected by staining with Bcl-2 BH3 antibody. E, Bax activation by BFC1108. Activated Bax was detected using Bax 6A7 antibody in tumor tissues from animals treated with vehicle or BFC1108. F, Activation of caspase-3 by BFC1108. Activated caspase-3 was detected using cleaved caspase-3 antibody in tumor tissues from animals treated with vehicle or BFC1108.</p

Supplemental Figure 1 from Identification and Characterization of a Small Molecule Bcl-2 Functional Converter

Bcl-2 dependent apoptotic effects of BFC1108. Independent experimental replicates of data in Figure 2C are shown.</p