Targeting of 6-Phosphofructo-2 kinase in Cancer

描述（由申请人提供）：肿瘤细胞在缺氧环境中生存和生长的能力需要一个全球性的转变，包括大量增加葡萄糖的使用，不仅用于能量活动，而且用于合成代谢活动。肿瘤细胞中的糖酵解通量由缺氧诱导因子1 （HIF-1）和致癌蛋白c-myc和ras激活。HIF-1和ras独立诱导果糖-2,6-二磷酸（F2,6BP）的合成，果糖-2,6-二磷酸是糖酵解的限速酶- 6-磷酸果糖-1激酶（PFK-1）的有效变构激活剂。F2,6BP的稳态浓度取决于6-磷酸果糖-2激酶（PFK-2）的活性，该酶在几种组织特异性亚型（PFKFB1-4）中表达。唯一在上皮细胞中表达的同种异构体，诱导型PFK-2 (iPFK-2; PFKFB3)，也被发现：(I)在人实体瘤原位过度表达，（ii）通过HIF-1a低氧暴露诱导；（iii） K562白血病肿瘤在体内生长所必需的。这项应用的长期目标是验证iPFK-2作为开发抗肿瘤药物的分子靶点。我们基于两个同源PFK-2同工酶的晶体结构建立了iPFK-2三级结构的计算模型，并筛选了iPFK-2结合位点的药效团匹配的虚拟组合文库。我们检测了81种得分较高的化合物对Jurkat T细胞白血病细胞的细胞毒活性，并鉴定了一种先导化合物F6P33，它可以抑制转化细胞（100 nM-1¿M）的糖酵解通量，并选择性地诱导细胞凋亡。我们现在提议保持沉默和

DESCRIPTION (provided by applicant): 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.