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Cancer-specific modification of 6-phosphofructo-1-kinase (PFK) impairs its ability to regulate the enzyme\u2019s activity which increases glycolytic flux. Consequently, excessive cytosolic NADH formation triggers a harmful redox imbalance in cancer cells, which is rapidly neutralized by the formation of lactic acid and superoxide (SOX). To learn more about deregulated glycolysis in cancer cells, a supercomputer used the atomic model of the crystal structure of human PFK1 for virtual screening a database of 4.5 million compounds by docking with the catalytic binding sites of the enzyme. The screening revealed two compounds capable of reducing modified, cancer-specific PFK1 activity and simultaneously suppressing lactate and SOX formation. A dose-dependent inhibition was observed in the cells treated by compounds in the following tumorigenic cells: Jurkat (Acute T cells leukemia); Caco-2 (colorectal adenocarcinoma); COLO 829 (melanoma); and MDA-MB-231 (breast gland adenocarcinoma). In addition, two selected compounds assessed for cytostatic and cytotoxic activity showed no negative effects on tumorigenic cells. However, during incubation, the strengths of inhibitions continuously decreased, both during lactate and SOX formation. No such effects were observed if compounds were sequentially submitted to the cells at low concentrations every 24 hours. Additional experiments performed by Jurkat cells revealed reduced respiration and glycolysis rates in the cells treated with compounds concerning the untreated cells. Inhibition of modified cancer-specific PFK1 activity reduces deregulated glycolytic flux, prevents abundant cytosolic NADH formation, and restores redox balance thus simultaneously preventing the formation of deleterious effects of lactate and SOX, two crucial players in cancer initiation and development.<\/jats:p>","DOI":"10.1371\/journal.pone.0321998","type":"journal-article","created":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T20:35:39Z","timestamp":1747859739000},"page":"e0321998","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":2,"title":["Small-molecule inhibitors of 6-phosphofructo-1-kinase simultaneously suppress lactate and superoxide generation in cancer 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