The ability of neoplastic cells to survive and grow in a hypoxic environment requires a global shift that involves large increases in the use of glucose, not only for energetic but also for anabolic activities. Glycolytic flux in neoplastic cells is activated by Hypoxia Inducible Factor 1 (HIF-1) and the oncogenic proteins c-myc and ras. HIF-1 and ras independently induce the synthesis of fructose-2,6-bisphosphate (F2,6BP) a potent allosteric activator of 6-phosphofructo-1-kinase (PFK-1), the rate-limiting enzyme of glycolysis. The steady-state concentration of F2,6BP depends on the activity of the enzyme 6-phosphofructo-2-kinase (PFK-2), which is expressed in several tissue-specific isoforms (PFKFB1-4). The only isoform expressed in epithelial cells, inducible PFK-2 (iPFK-2; PFKFB3), also has been found to be: (I) over-expressed by human solid tumors in situ, (ii) induced by hypoxic exposure via HIF-1a; and (iii) required for K562 leukemia tumor growth in vivo. The long-term objectives of this application are to validate iPFK-2 as a molecular target for the development of anti-neoplastic agents. We developed a computational model of the tertiary structure of iPFK-2 based on the crystal structures of two homologous PFK-2 isozymes and screened virtual combinatorial libraries for pharmacophore fits of the fructose-6-phosphate (F6P) binding site of iPFK-2. We examined 81 highly scored compounds for cytotoxic activity against Jurkat T cell leukemia cells and identified a lead compound, designated F6P33, that suppresses glycolytic flux to lactate and induces apoptosis selectively in transformed cells (100 nM-1?M). We now propose to both silence and ? ectopically enhance iPFK-2 in transformed cells in vitro and in vivo and examine the consequences on the anti-neoplastic effects of F6P33. We expect that these studies will support the role of iPFK-2 as the molecular target of F6P33 and thus validate the development of small molecule inhibitors of iPFK-2 as antineoplastic agents. ? ?
