Inhibition of Angiogenesis by TNP 470 and Ovalicin

描述(申请人提供)：蛋白质合成过程中起始物质蛋氨酸的翻译后加工是一种从原核生物到真核生物保守的普遍生物学过程。蛋氨酸氨基肽酶是催化脱除引发剂蛋氨酸的酶。一个基因在原核生物中已知，而两个基因在真核生物中已知，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都是致命的，这一事实突显了启动蛋氨酸加工的重要性。在真核生物的两个蛋氨酸氨基肽酶基因中，2型酶(MetAP2)已被证明是烟青素血管生成抑制剂家族的直接靶点，包括其类似物TNP-470。过去几年的研究提供了强有力的证据，表明MetAP2是TNP-470的一个生理相关靶点。研究还发现，TNP-470抑制内皮细胞增殖是由肿瘤抑制基因P53介导的。因此，TNP-470能够激活P53，从而诱导p21的表达，p21负责对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，一种新的抗癌药物苯乙酰胺被发现在体外同时抑制MetAP2和MetAP1，并将细胞周期阻断在G1和G2/M期。推测MetAP1可能在G2/M期细胞周期中发挥作用。本研究的主要目的是以酵母为模型系统，进一步阐明MetAP1和MetAP2的生理功能，并通过高通量筛选筛选出MetAP1和MetAP2的异构体特异性抑制物。通过建立表达MetAP1和MetAP2不同结构域的酵母突变体，并利用DNA芯片检测表型变化，来研究MetAP1和MetAP2中不同结构域的功能。我们将系统地研究已知的不同的P53激活剂，以确定TNP-470激活P53的潜在介质。将制备苯乙酰胺的分子探针，以确认其与MetAP1和MetAP2的相互作用。将进行高通量筛选，以确定MetAP1和MetAP2的特定抑制剂。新发现的MetAP1抑制剂将被用来评估MetAP1在G2/M期细胞周期进展中的生理作用。

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.