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 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: The following primary and secondary antibodies were used: ENO1 (Abcam, # ab227978; 1:10000), ENO3 (Novus Biologicals, # NBP1-31764; 1:2000) β-actin (CST, #4970S; 1:10000), GAPDH (CST, #2118S; 1:10000), β-catenin (CST, #8480T; 1:1000), Lamin B1 (CST, #13435S; 1:1000), COX4 (CST, #38563; 1:1000), anti-mouse IgG HRP-linked antibody (CST, #7076, 1:5000), and anti-rabbit IgG HRP-linked antibody (CST, #7074, 1:5000).

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