Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 targets ENO1 (A) Chemical structure of podophyllotoxin (parental compound) and SU212. (B–D) SU212 binds to ENO1 and ENO3. Target identification using CETSA: Differential profiling of SU212 on the thermal proteome profile of MDA-MB-231 cells. Cells were treated with DMSO or SU212 (0.5 μM) for 1.5 h and then lysed, and an equal quantity of soluble protein was labeled with a tandem mass tag, followed by liquid chromatography-tandem mass spectrometry analysis. (B) Heatmap representation of the thermal stability of 1,074 soluble proteins in cancer cells treated with vehicle-DMSO (left) and SU212 (right). (C) A scatterplot of melting temperature (T m ) calculated after SU212 and vehicle treatment. Proteins that passed the significant value thresholds ( p < 0.01, R2 > 0.8) and identification criteria are highlighted in orange. (D) Melting curves for ENO1/ENO3 with and without SU212 treatment depict the change in T m . (E) Representative sensorgrams for ENO1/3-SU212 interaction. His-tagged ENO1 and ENO3 proteins were immobilized on a Ni-NTA sensor, and SU212 (10 μM) was tested for physical interaction using BLI. (F) SU212 physically interacts with ENO1 protein. The BLI sensorgrams were obtained using His tag-ENO1-loaded Octet NTA biosensors and SU212 (1, 5, 10 μM). (G) SU212 treatment inhibits ENO1 expression. Immunoblotting: TNBC cells were treated with vehicle only (DMSO) and SU212 (0.1, 0.25, 0.5 μM) for 6 h. SDS-PAGE and western blot analysis were performed for the ENO1 and ENO3 proteins. Membranes were stripped and re-probed with anti-beta-actin antibody to ensure equal protein loading. (H) SU212 treatment leads to degradation of the ENO1 protein. Immunoblotting: MDA-MB-231 cells were treated with combinations of SU212, CHX, MG132, 3MA, and NH4Cl, as depicted in the figure, for 6 h. SDS-PAGE and western blot analysis were performed for the ENO1 protein. Membranes were stripped and re-probed with anti-GAPDH antibody to ensure equal protein loading. (I) SU212 treatment induces apoptotic cell death in MDA-MB-231 cells. Data are shown as the mean ± SD ( n = 5). Numbers indicate a p value compared with the vehicle control and analyzed using two-way ANOVA. (J) Enolase enzyme activity assay. PC, positive control. Data are shown as the mean ± SD ( n = 3). Numbers indicate a p value that is different compared with vehicle control, analyzed using Student’s t test. ns, not significant.

Article Snippet: During the loading step, recombinant His Tag-Enolases were immobilized on NTA Biosensors using a kinetic buffer (1X PBS) with a final reagent volume of 50 μL, which contained 50 μg/mL of ENO1 (#11554-H07E−100, Sino Biological) and ENO3 (#14270-H07E, Sino Biological), all placed in a black 384-well microplate.

Techniques: Drug discovery, Labeling, Liquid Chromatography, Mass Spectrometry, Expressing, Western Blot, SDS Page, Control, Enzyme Activity Assay, Positive Control

Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 modulates the ENO1-associated pathways (A) Seahorse XF glycolytic rate assay on TNBC cells (left), normal mammary epithelial cells (middle), or HEK293 cells (right). Data are shown as the mean ± SD ( n = 6). Normalized proton efflux rate (PER) data were analyzed using two-way ANOVA. (B–D) SU212 treatment inhibits the FDG (glucose analog) uptake by tumor cells. Female NSG mice subcutaneously implanted with MDA-MB-231 cells. Mice were treated with vehicle and SU212 (30 mg/kg) for 3 days prior to PET scan; mice were injected with 200 μCi F18-FDG and imaged using microPET/CT system. Data are shown as the mean ± SD ( n = 6). Numbers indicate a p value compared with the vehicle control, analyzed using two-way ANOVA. (B) Representative PET scan images. (C) Tumors were collected at the end of the PET scan, and tumor weights were collected. (D) PET signal quantification for F18-FDG accumulation in the tumor region. Data are shown as the mean ± SD ( n = 5). Numbers indicate a p value that is different compared with vehicle control, analyzed using Student’s t test. (E and F) SU212 treatment inhibits the mTORC1 pathway in TNBC cells. Gene set enrichment analysis was performed on the proteomics results to determine the enrichment of KEGG pathways upon treatment of MDA-MB-231, MDA-MB-468, and BT549 cell lines with SU212 (6 hr, 0.5 μM). (E) Enrichment analysis of downregulated proteins. (F) Enrichment analysis of upregulated proteins.

Article Snippet: During the loading step, recombinant His Tag-Enolases were immobilized on NTA Biosensors using a kinetic buffer (1X PBS) with a final reagent volume of 50 μL, which contained 50 μg/mL of ENO1 (#11554-H07E−100, Sino Biological) and ENO3 (#14270-H07E, Sino Biological), all placed in a black 384-well microplate.

Techniques: Injection, Control

Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 inhibits ENO1 cellular localization and TNBC migration and metastasis (A–C) SU212 inhibits ENO1 cellular localization. MDA-MB-231 (top row) and EMT6 (bottom row) cells were treated with DMSO and SU212 (0.1 μM) for 6 h, and cells were fractionated for different cellular compartments. SDS-PAGE and western blot analysis were performed to identify ENO1 protein. Membranes were stripped and re-probed with respective loading control antibodies to ensure equal protein loading. (A) Cell membrane and cytosolic protein fractionation. β-catenin identifies the membrane fraction. (B) Cell nucleus and cytosolic protein fractionation. Lamin B1 identifies the nuclear fraction. (C) Mitochondrial and cytosolic protein fractionation. COX4 identifies the mitochondrial fraction. (D) MG132 and 3MA co-treatment partially reverses the SU212-associated inhibition of ENO1 cell membrane localization. (E) Matrigel dot invasion assay demonstrates SU212 treatment inhibits cell invasion and migration in TNBC cells. The data shown are mean ± SD ( n = 3). Data were analyzed using the Student’s t test. Numbers indicate a p value that is different compared with vehicle control. (F–H) SU212 treatment inhibits lung metastasis. In total, 1 × 10 5 EMT6 cells were implanted orthotopically in female NSG mice. Mice were randomized and divided into two groups. Each group was treated with either vehicle only or SU212 (30 mg/kg) for 21 days. Lungs were collected and processed for H&E staining. (F) Representative lung images collected at experiment end. (G) Representative images of H&E-stained lungs. Scale bar, 1,000 μm. (H) Number of metastatic foci. Data are shown as the mean ± SD ( n = 7/group). Numbers indicate the p value compared with vehicle control; data analyzed using Student’s t test.

Article Snippet: During the loading step, recombinant His Tag-Enolases were immobilized on NTA Biosensors using a kinetic buffer (1X PBS) with a final reagent volume of 50 μL, which contained 50 μg/mL of ENO1 (#11554-H07E−100, Sino Biological) and ENO3 (#14270-H07E, Sino Biological), all placed in a black 384-well microplate.

Techniques: Migration, SDS Page, Western Blot, Control, Membrane, Fractionation, Inhibition, Invasion Assay, Staining

Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 therapy inhibits tumor growth in a diabetic mouse model and improves fatty liver conditions In total, 1 × 10 5 PY8119 cells were implanted orthotopically in female Db/Db (B6.BKS(D)-Leprdb/J, homozygous for Lepr) mice. Mice were randomized and divided into two groups (vehicle control n = 9, treatment n = 10). Each group was treated with vehicle only or SU212 (10 mg/kg) for 32 days, i.p. (A) Image of representative tumors collected at the end of treatment. (B) Tumor volume was measured over time. (C) Tumor weight collected at the end of the study. (D and E) (D) Representative pictographs of ENO1 immunohistochemistry (IHC) staining of tumor (×20 magnification; scale bar, 50 μm). (E) Mouse body weight was measured over time. (F) Blood glucose measured over time. (G) Blood glucose was measured at day 0 and 1 post-treatment. (H) Blood samples were collected from each mouse at the end of treatment. The serum was analyzed for levels of liver (AST, ALT, ALP, GGT, and GLDH) health biochemical markers on a Siemens Dimension Xpand Plus HM analyzer. (I and J) At the end of treatment, livers were collected and processed for H&E staining. (I) Liver weight collected at the end of the study. (J) Representative pictographs of H&E staining of liver (×20 magnification; scale bar, 50 μm). (K and L) (K) Percentage fat-associated space scored in H&E-stained liver slides. (L) Quantitative gene expression analysis. At the end of treatment, RNA was isolated from each tumor sample and processed for gene expression analysis using NanoString nCounter Mouse Metabolic Pathways Panels. Data represent gene expression for respective pathways. All data are shown as the mean ± SD ( n = 5/group). Numbers indicate the p value compared with vehicle control. The data were analyzed using either Student’s t test (C, D, G, H, J, and L) or two-way ANOVA (B, E, and F).

Article Snippet: During the loading step, recombinant His Tag-Enolases were immobilized on NTA Biosensors using a kinetic buffer (1X PBS) with a final reagent volume of 50 μL, which contained 50 μg/mL of ENO1 (#11554-H07E−100, Sino Biological) and ENO3 (#14270-H07E, Sino Biological), all placed in a black 384-well microplate.

Techniques: Control, Immunohistochemistry, Staining, Gene Expression, Isolation

Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 targets ENO1 (A) Chemical structure of podophyllotoxin (parental compound) and SU212. (B–D) SU212 binds to ENO1 and ENO3. Target identification using CETSA: Differential profiling of SU212 on the thermal proteome profile of MDA-MB-231 cells. Cells were treated with DMSO or SU212 (0.5 μM) for 1.5 h and then lysed, and an equal quantity of soluble protein was labeled with a tandem mass tag, followed by liquid chromatography-tandem mass spectrometry analysis. (B) Heatmap representation of the thermal stability of 1,074 soluble proteins in cancer cells treated with vehicle-DMSO (left) and SU212 (right). (C) A scatterplot of melting temperature (T m ) calculated after SU212 and vehicle treatment. Proteins that passed the significant value thresholds ( p < 0.01, R2 > 0.8) and identification criteria are highlighted in orange. (D) Melting curves for ENO1/ENO3 with and without SU212 treatment depict the change in T m . (E) Representative sensorgrams for ENO1/3-SU212 interaction. His-tagged ENO1 and ENO3 proteins were immobilized on a Ni-NTA sensor, and SU212 (10 μM) was tested for physical interaction using BLI. (F) SU212 physically interacts with ENO1 protein. The BLI sensorgrams were obtained using His tag-ENO1-loaded Octet NTA biosensors and SU212 (1, 5, 10 μM). (G) SU212 treatment inhibits ENO1 expression. Immunoblotting: TNBC cells were treated with vehicle only (DMSO) and SU212 (0.1, 0.25, 0.5 μM) for 6 h. SDS-PAGE and western blot analysis were performed for the ENO1 and ENO3 proteins. Membranes were stripped and re-probed with anti-beta-actin antibody to ensure equal protein loading. (H) SU212 treatment leads to degradation of the ENO1 protein. Immunoblotting: MDA-MB-231 cells were treated with combinations of SU212, CHX, MG132, 3MA, and NH4Cl, as depicted in the figure, for 6 h. SDS-PAGE and western blot analysis were performed for the ENO1 protein. Membranes were stripped and re-probed with anti-GAPDH antibody to ensure equal protein loading. (I) SU212 treatment induces apoptotic cell death in MDA-MB-231 cells. Data are shown as the mean ± SD ( n = 5). Numbers indicate a p value compared with the vehicle control and analyzed using two-way ANOVA. (J) Enolase enzyme activity assay. PC, positive control. Data are shown as the mean ± SD ( n = 3). Numbers indicate a p value that is different compared with vehicle control, analyzed using Student’s t test. ns, not significant.

Article Snippet: ENO1 protein , Sino Biological , Cat. No. # 11554-H07E−100.

Techniques: Drug discovery, Labeling, Liquid Chromatography, Mass Spectrometry, Expressing, Western Blot, SDS Page, Control, Enzyme Activity Assay, Positive Control

Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 modulates the ENO1-associated pathways (A) Seahorse XF glycolytic rate assay on TNBC cells (left), normal mammary epithelial cells (middle), or HEK293 cells (right). Data are shown as the mean ± SD ( n = 6). Normalized proton efflux rate (PER) data were analyzed using two-way ANOVA. (B–D) SU212 treatment inhibits the FDG (glucose analog) uptake by tumor cells. Female NSG mice subcutaneously implanted with MDA-MB-231 cells. Mice were treated with vehicle and SU212 (30 mg/kg) for 3 days prior to PET scan; mice were injected with 200 μCi F18-FDG and imaged using microPET/CT system. Data are shown as the mean ± SD ( n = 6). Numbers indicate a p value compared with the vehicle control, analyzed using two-way ANOVA. (B) Representative PET scan images. (C) Tumors were collected at the end of the PET scan, and tumor weights were collected. (D) PET signal quantification for F18-FDG accumulation in the tumor region. Data are shown as the mean ± SD ( n = 5). Numbers indicate a p value that is different compared with vehicle control, analyzed using Student’s t test. (E and F) SU212 treatment inhibits the mTORC1 pathway in TNBC cells. Gene set enrichment analysis was performed on the proteomics results to determine the enrichment of KEGG pathways upon treatment of MDA-MB-231, MDA-MB-468, and BT549 cell lines with SU212 (6 hr, 0.5 μM). (E) Enrichment analysis of downregulated proteins. (F) Enrichment analysis of upregulated proteins.

Article Snippet: ENO1 protein , Sino Biological , Cat. No. # 11554-H07E−100.

Techniques: Injection, Control

Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 inhibits ENO1 cellular localization and TNBC migration and metastasis (A–C) SU212 inhibits ENO1 cellular localization. MDA-MB-231 (top row) and EMT6 (bottom row) cells were treated with DMSO and SU212 (0.1 μM) for 6 h, and cells were fractionated for different cellular compartments. SDS-PAGE and western blot analysis were performed to identify ENO1 protein. Membranes were stripped and re-probed with respective loading control antibodies to ensure equal protein loading. (A) Cell membrane and cytosolic protein fractionation. β-catenin identifies the membrane fraction. (B) Cell nucleus and cytosolic protein fractionation. Lamin B1 identifies the nuclear fraction. (C) Mitochondrial and cytosolic protein fractionation. COX4 identifies the mitochondrial fraction. (D) MG132 and 3MA co-treatment partially reverses the SU212-associated inhibition of ENO1 cell membrane localization. (E) Matrigel dot invasion assay demonstrates SU212 treatment inhibits cell invasion and migration in TNBC cells. The data shown are mean ± SD ( n = 3). Data were analyzed using the Student’s t test. Numbers indicate a p value that is different compared with vehicle control. (F–H) SU212 treatment inhibits lung metastasis. In total, 1 × 10 5 EMT6 cells were implanted orthotopically in female NSG mice. Mice were randomized and divided into two groups. Each group was treated with either vehicle only or SU212 (30 mg/kg) for 21 days. Lungs were collected and processed for H&E staining. (F) Representative lung images collected at experiment end. (G) Representative images of H&E-stained lungs. Scale bar, 1,000 μm. (H) Number of metastatic foci. Data are shown as the mean ± SD ( n = 7/group). Numbers indicate the p value compared with vehicle control; data analyzed using Student’s t test.

Article Snippet: ENO1 protein , Sino Biological , Cat. No. # 11554-H07E−100.

Techniques: Migration, SDS Page, Western Blot, Control, Membrane, Fractionation, Inhibition, Invasion Assay, Staining

Journal: Cell Reports Medicine

Article Title: Non-orthosteric inhibition of enolase 1 impedes growth of triple-negative breast cancer

doi: 10.1016/j.xcrm.2025.102451

Figure Lengend Snippet: SU212 therapy inhibits tumor growth in a diabetic mouse model and improves fatty liver conditions In total, 1 × 10 5 PY8119 cells were implanted orthotopically in female Db/Db (B6.BKS(D)-Leprdb/J, homozygous for Lepr) mice. Mice were randomized and divided into two groups (vehicle control n = 9, treatment n = 10). Each group was treated with vehicle only or SU212 (10 mg/kg) for 32 days, i.p. (A) Image of representative tumors collected at the end of treatment. (B) Tumor volume was measured over time. (C) Tumor weight collected at the end of the study. (D and E) (D) Representative pictographs of ENO1 immunohistochemistry (IHC) staining of tumor (×20 magnification; scale bar, 50 μm). (E) Mouse body weight was measured over time. (F) Blood glucose measured over time. (G) Blood glucose was measured at day 0 and 1 post-treatment. (H) Blood samples were collected from each mouse at the end of treatment. The serum was analyzed for levels of liver (AST, ALT, ALP, GGT, and GLDH) health biochemical markers on a Siemens Dimension Xpand Plus HM analyzer. (I and J) At the end of treatment, livers were collected and processed for H&E staining. (I) Liver weight collected at the end of the study. (J) Representative pictographs of H&E staining of liver (×20 magnification; scale bar, 50 μm). (K and L) (K) Percentage fat-associated space scored in H&E-stained liver slides. (L) Quantitative gene expression analysis. At the end of treatment, RNA was isolated from each tumor sample and processed for gene expression analysis using NanoString nCounter Mouse Metabolic Pathways Panels. Data represent gene expression for respective pathways. All data are shown as the mean ± SD ( n = 5/group). Numbers indicate the p value compared with vehicle control. The data were analyzed using either Student’s t test (C, D, G, H, J, and L) or two-way ANOVA (B, E, and F).

Article Snippet: ENO1 protein , Sino Biological , Cat. No. # 11554-H07E−100.

Techniques: Control, Immunohistochemistry, Staining, Gene Expression, Isolation