Inhibition of Angiogenesis by TNP 470 and Ovalicin

TNP 470 和 Ovalicin 抑制血管生成

• 批准号: 6893356
• 负责人: Jun O. Liu
• 金额: $ 38.42万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-05 至 2008-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6893356
• 关键词: SDS polyacrylamide gel electrophoresis; aminopeptidase; angiogenesis inhibitors; antineoplastics; azepines; cell cycle; drug screening /evaluation; enzyme inhibitors; enzyme mechanism; fungal genetics; gene expression; high throughput technology; microarray technology; p53 gene /protein; protein binding; protein protein interaction; tissue /cell culture; yeasts

DESCRIPTION (provided by applicant): The post-translational processing of initiator methionine during protein synthesis is a universal biological process that is conserved from prokaryotes to eukaryotes. Methionine aminopeptidases are the enzymes catalyzing the removal of initiator methionine. One gene is known in prokaryotes while two genes are known in eukaryotes encoding methionine aminopeptidases. The importance of initiator methionine processing is underscored by the fact that deletion of the methionine aminopeptidase genes in either prokaryotes or eukaryotes is lethal. Of the two methionine aminopeptidase genes in eukaryotes, the type 2 enzyme (MetAP2) has been shown to be the direct target for the fumagillin family of angiogenesis inhibitors, including its analog TNP-470. Work in the past several years has provided strong evidence that MetAP2 is a physiologically relevant target for TNP-470. It has also been found that inhibition of endothelial cell proliferation by TNP-470 is mediated by the tumor suppressor gene p53. Thus, TNP-470 is capable of activating p53, which induces the expression of p21that is responsible for the cell cycle blockade of endothelial cells. These studies reveal a unique role of MetAP2 in the progression of the endothelial cell cycle. Recently, a novel anticancer drug entering Phase II clinical trial known as bengamide was found to inhibit both MetAP2 and MetAP1 in vitro and to block cell cycle in both G1 and G2/M phase. It is hypothesized that MetAP1 may play a role in the cell cycle at the G2/M phase. The major objective of the current proposal is to further delineate the physiological functions of MetAP1 and MetAP2 employing yeast as a model system and to identify isoform-specific inhibitors for MetAP1 and MetAP2 by high throughput screens. The functions of the different domains in MetAP1 and MetAP2 will be investigated by creating various yeast mutants expressing different domains of these enzymes and determining the phenotypic changes using DNA microarray. The different known activators of p53 will be systematically examined to identify potential mediators of p53 activation by TNP-470. Molecular probes of bengamide will be prepared to confirm its interaction with MetAP1 and MetAP2. High throughput screens will be conducted to identify specific inhibitors for MetAP1 and MetAP2. The newly identified inhibitors for MetAP1 will be employed to assess the physiological role of MetAP1 in the cell cycle progression in the G2/M phase.

描述（由申请人提供）：蛋白质合成过程中引发剂蛋氨酸的翻译后加工是一个普遍的生物过程，从原核生物到真核生物都是保守的。蛋氨酸氨基肽酶是催化脱除引发剂蛋氨酸的酶。在原核生物中已知一个基因，而在真核生物中已知两个基因编码蛋氨酸氨基肽酶。在原核生物或真核生物中，蛋氨酸氨基肽酶基因的缺失是致命的，这一事实强调了引发剂蛋氨酸加工的重要性。在真核生物的两种蛋氨酸氨基肽酶基因中，2型酶（MetAP2）已被证明是富马青霉素家族血管生成抑制剂的直接靶点，包括其类似物TNP-470。过去几年的工作提供了强有力的证据，证明MetAP2是TNP-470的生理相关靶点。研究还发现，TNP-470对内皮细胞增殖的抑制是由肿瘤抑制基因p53介导的。因此，TNP-470能够激活p53，从而诱导p21的表达，而p21负责内皮细胞的细胞周期阻断。这些研究揭示了MetAP2在内皮细胞周期进程中的独特作用。最近，一种进入II期临床试验的新型抗癌药物bengamide被发现在体外抑制MetAP2和MetAP1，并在G1和G2/M期阻断细胞周期。我们推测MetAP1可能在细胞周期G2/M期发挥作用。目前的主要目标是利用酵母作为模型系统进一步描述MetAP1和MetAP2的生理功能，并通过高通量筛选鉴定MetAP1和MetAP2的异构体特异性抑制剂。MetAP1和MetAP2中不同结构域的功能将通过创建表达这些酶不同结构域的各种酵母突变体和使用DNA微阵列确定表型变化来研究。我们将系统地研究不同的已知p53激活因子，以确定TNP-470激活p53的潜在介质。制备bengamide分子探针，确认其与MetAP1和MetAP2的相互作用。将进行高通量筛选，以确定MetAP1和MetAP2的特异性抑制剂。新发现的MetAP1抑制剂将用于评估MetAP1在G2/M期细胞周期进程中的生理作用。