Inhibiting Multi-Functional ALDOA for Cancer Therapy

果糖二磷酸醛缩酶A（ALDOA）是一个古老的，高表达的基因，已获得三个 不同的细胞活动。最好理解的活性是催化有氧糖酵解的关键步骤。ALDOA 也具有不依赖于其催化活性的蛋白质结合活性（“兼职”），包括结合 细胞骨架蛋白，其使用适合ALDO的“兼职口袋”的“EΦE”基序。结合至 细胞骨架肌动蛋白将ALDO保持在非活性状态，直到需要时，细胞骨架肌动蛋白的增加会释放ALDO。 其底物果糖-1，6-二磷酸水平和/或生长因子诱导的PI-3-激酶活性。另一 ALDOA的兼职功能是它存在于癌细胞的细胞核中，在那里它与 增加扩散。一种可能性是，核ALDOA结合到核中EID-1上的EΦE基序 抑制HIF-1的转录辅激活因子p300。ALDOA水平在许多癌症中增加，特别是 胰腺癌，它与患者生存率低和转移增加有关。胰腺 癌细胞具有高水平的无氧糖酵解，其通过缺氧诱导的糖酵解进一步增加。 转录因子-1（HIF-1）。HIF-1诱导ALDOA和其他糖酵解酶，这些酶反过来维持高水平的糖酵解。 HIF-1活性通过AMPK/p300依赖性前馈环。HIF-1活性导致VEGF释放， 血管生成和诱导其他癌细胞存活机制。糖酵解的增加提供了 低氧癌细胞具有增加的能量供应（ATP）和生物质必需代谢物 合成. ALDOA升高还与癌细胞紧密连接的一种成分E-钙粘蛋白降低有关 (TJ)细胞间相互作用所必需的，给予更多的间充质表型和增加的肿瘤 转移这很可能是由于ALDOA与细胞骨架肌动蛋白的EΦE基序结合，导致其 聚合和分解，或其他失活的TJ的E-钙粘蛋白的损失。这一切使得ALDOA 一种特殊的可药用抗癌靶点。我们的X射线晶体学研究已经确定了一个新的作用， ALDO的C-末端结构域在其催化活性中，以及反应性Cys 289残基， 催化活性的变构调节。我们已经确定了一种新的铅探针ALDOA变构抑制剂 与Cys 289形成复合物，抑制糖酵解、HIF-1活性和癌细胞增殖， 在肿瘤异种移植模型中体内抑制糖酵解和肿瘤生长。利用这种生物稳定的变构物质 抑制剂作为先导，我们的目标之一是制造更有效的抑制剂，结合到变构位点Cys 289， 以及同时接合活性位点和兼职口袋的高亲和力双功能抑制剂。 我们将使用X射线晶体学来帮助我们设计抑制剂，调节HIF-1活性的核调节因子; 尤其是设计ALDOA催化活性的直接抑制剂。我们将使用这些化合物作为 药理学探针，以抑制ALDOA的各种活动，并作为潜在的领导新的治疗方法， 胰腺癌和其他癌症的治疗。

Fructose-bisphosphate aldolase A (ALDOA) is an ancient, highly expressed gene that has acquired three distinct cellular activities. The best understood activity is catalyzing a key step in aerobic glycolysis. ALDOA also has protein binding activities independent of its catalytic activity (“moonlighting”), that include binding to cytoskeletal proteins, which use an “EΦE” motif that fits into a “moonlighting pocket” of ALDO. Binding to the cytoskeleton actin holds ALDO in an inactive form until it is needed, when it is released by an increase in the levels of its substrate fructose-1, 6-bisphosphate and/or by growth-factor-induced PI-3-kinase activity. Another moonlighting function of ALDOA is its presence in the nucleus of cancer cells, where it is associated with increased proliferation. One possibility is that nuclear ALDOA binds to the EΦE motif on EID-1 in the nucleus to inhibit HIF-1's transcriptional co-activator, p300. ALDOA levels are increased in many cancers, particularly pancreatic cancer, where it has been linked to poor patient survival and an increase in metastasis. Pancreatic cancer cell has high levels of anaerobic glycolysis that is further increased by the hypoxia inducible transcription factor-1 (HIF-1). HIF-1 induces ALDOA and other glycolytic enzymes that in turn maintain high HIF-1 activity through an AMPK/p300-dependent feed-forward loop. HIF-1 activity leads to VEGF release and angiogenesis, and the induction of other cancer cell survival mechanisms. Increased glycolysis provides hypoxic cancer cells with an increased supply of energy (ATP) and essential metabolites for biomass synthesis. Elevated ALDOA is also associated with low E-cadherin, a component of cancer cell tight junctions (TJ) necessary for cell-cell interactions, giving a more mesenchymal phenotype and increased tumor metastasis. This most likely is due to binding of ALDOA to the EΦE motif of cytoskeleton actin causing its polymerization and disassembly, or other inactivation of the TJ with loss of E-cadherin. All this makes ALDOA an exceptional druggable anti-cancer target. Our X-ray crystallography studies have identified a new role for the C-terminal domain of ALDO in its catalytic activity, as well as a reactive Cys289 residue that allows allosteric regulation of catalytic activity. We have identified a novel lead probe allosteric inhibitor of ALDOA that forms a complex with Cys289 inhibiting glycolysis, HIF-1 activity and the proliferation of cancer cells, and in vivo inhibits glycolysis and tumor growth in a tumor xenograft model. Using this biologically stable allosteric inhibitor as a lead, one of our goals is to make more potent inhibitors that bind to the allosteric site Cys289, as well as high affinity bifunctional inhibitors that simultaneously engage the active site and moonlighting pocket. We will use X-ray crystallography to help us design inhibitors that modulate nuclear regulators of HIF-1 activity; and not least to design direct inhibitors of ALDOA catalytic activity. We will use these compounds as pharmacological probes to inhibit the various activities of ALDOA, and as potential leads for new therapies for the treatment of pancreatic, and other cancers.