Bioenergetic and proteomic profiling to screen small molecule inhibitors that target cancer metabolisms

A novel inhibitor of tumorspheres reveals the activation of the serine biosynthetic pathway upon mitochondrial inhibition

“…2E). This pattern is similar to that of the complex III inhibitor antimycin A [16], which indicates that NPD2381 should inhibit complex III activity. Unexpectedly, when we examined the effect of NPD2381 on the activity of mitochondrial complexes, we found that it inhibited the activity of complex I and II, but only weakly inhibited complex III ( Fig.…”

“…Hence, increased serine biosynthesis could be a general cytoprotective and compensatory metabolic adaptation that counters the therapeutic efficacy of mitochondrial inhibitors. The proliferation of HeLa or HCT116 cells was not The data for oligomycin A, piericidin A and antimycin A in glycolysis column have been already reported elsewhere [16], but are shown here for comparison. The inhibitors were used at a concentration of 10 lM for 18 h. (B) HCT116 and HeLa cells treated with NPD2381(10 lM) were analysed for PHGDH expression by western blot analysis.…”

“…The scores indicate the integrated value of selected bars (coloured red). The data for oligomycin A, piericidin A and antimycin A in glycolysis column have been already reported elsewhere , but are shown here for comparison. The inhibitors were used at a concentration of 10 μ m for 18 h. (B) HCT 116 and HeLa cells treated with NPD 2381(10 μ m ) were analysed for PHGDH expression by western blot analysis.…”

“…Gel images were analysed with ProgenesisSameSpots (Total Lab, Newcastle, UK). Of the more than 1000 spots detected in each 2D gel, 296 individual spots were found to be in common between the gels of the reference and compound‐treated cells, and were selected as previously described . Next, the volume of each spot was normalized using the average of the corresponding control values from DMSO‐treated HeLa cells, and the log‐fold ratios of the normalized volumes and the average values from the triplicates were calculated.…”

Identification of a Small Compound Targeting PKM2-Regulated Signaling Using 2D Gel Electrophoresis-Based Proteome-wide CETSA

“…A database of 2D DIGE-based proteomics, ChemProteoBase, including profiles of proteomic perturbation induced by the target-validated compounds (Muroi et al, 2010;Muroi and Osada, 2019) was constructed to compare similarities in protein expression patterns induced by each of the target-validated compounds and to predict the mechanism of action of new bioactive compounds (Kawamura et al, 2016;Kawatani et al, 2011Kawatani et al, , 2016Muroi et al, 2010). We previously identified 296 proteins in ChemProteoBase that appeared as protein spots on the 2D gel of cervical cancer HeLa cell lysates and created a map of original protein spots linking the protein information (Futamura et al, 2019). This accumulated knowledge and resources will be helpful for the identification of proteins with altered thermal stability detected by the 2DE-CETSA.…”