Post-translational modification of the 6-phosphofructo-1-kinase (PFK-1) in cancers results in the formation of highly-active enzyme that might be the pivotal factor of deregulated glycolytic flux and consequently lactate generation and excretion. Human cells express three different PFK1 isozymes: muscle type (PFK-M), platelet type (PFK-P), and liver type (PFK-L). At the National Institute of Chemistry in Ljubljana, sixteen low-molecular compounds were virtually selected by a supercomputer that might bind to the to the ATP-binding of the 3-D crystal structure of human PFK-L enzyme. My goal was to test the selected compounds for preventing lactate generation by inhibiting PFK-L activity in three selected tumorigenic cell lines. Preliminary tests revealed the strongest inhibition of lactate formation by inhibitors designated 23, 29, 30, 31, and 32. By more detailed dose dependent experiments, the efficiencies of individual inhibitors in specific tumorigenic cell lines were estimated. The obtained results showed different reduction of lactate formation by selected inhibitors at individual cell lines. Because the reductions in the strengths of inhibitions were occasionally observed during the prolonged 90 hours incubation, sequential addition of the inhibitors has been tested. By repeated addition of inhibitors at low (10-30 µM) concentrations at 24 hours intervals, lactate generation was largely prevented in all tested tumorigenic cell lines. Due to tissue-specific occurrence of isozymes, we also tested a combination of first- and second-generation inhibitors. Lower concentrations of the lactate were recorded already at the lowest concentrations of inhibitors (20 µM). Based on these results, it can be concluded that discovered small-molecule-inhibitors can decrease the deregulated glycolytic flux the cancer cells and effectively prevent lactate generation. The selected inhibitors might in combination with other therapeutic methods help to prevent the growth of different tumors.
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