INHIBITION OF ANGIOGENESIS BY TNP 470 AND OVALICIN

描述：本提案的主要目的是获得一种分子 了解天然血管生成抑制机制， 产品烟曲霉素、卵青霉素和合成类似物TNP-470，以及 通过靶向治疗，提高疗效，减少副作用 他们的肿瘤。 最近的研究表明，TNP-470和卵蒜素 有效抑制2型甲硫氨酸的酶活性 氨肽酶（MetAP 2），这表明MetAP 2是一种特异性靶点， TNP-470和卵蒜素。 来描述分子间的相互作用 药物和MetAP 2，药物的酶结合形式和药物中的残留物， 将鉴定被药物共价修饰的MetAP 2， 表征了 生成随机MetAP 2突变体文库并进行 基因筛选将鉴定来自MetAP 2的额外残基， 与药物结合有关。 确认MetAP 2是目标 内皮细胞中的药物，包括野生型和耐药MetAP 2突变体 将在内皮细胞中表达，以检查它们是否赋予 内皮细胞对药物的耐药性。 两个最明显的 抑制MetAP 2的作用是阻断N-末端豆蔻酰化， MetAP 2底物之间蛋白质周转率的改变， 这可以解释内皮细胞生长的抑制， 毒品 为了鉴定相关的MetAP 2底物，将检测蛋白质 并通过以下方式鉴定其肉豆蔻酰化或转换受药物影响： 用（3 H）-肉豆蔻酸和（35 S）-甲硫氨酸标记全细胞蛋白。 一旦这种MetAP 2底物被鉴定，它们在介导代谢中的作用就被阐明了。 将在内皮细胞中进一步研究药物的作用。 的 最终目标将是实现一个完整的了解机制， 通过鉴定药物对内皮细胞生长的抑制 药物作用的直接和间接介质。 最后， 烟曲霉素和卵蒜素将与环状RGD肽缀合， 将药物靶向肿瘤部位的内皮细胞， 表达高水平的RGD结合α-v-β-3整联蛋白。 这些 药物-RGD肽缀合物可能被证明具有更高的选择性， 比TNP-470和相关的血管生成抑制剂更毒。

DESCRIPTION: The main objective of this proposal is to gain a molecular understanding of the mechanism of inhibition of angiogenesis by the natural products fumagillin, ovalicin and the synthetic analog TNP-470, and to improve the efficacy and reduce the side effects of these drugs by targeting them to tumors. It was recently shown that both TNP-470 and ovalicin potently inhibit the enzymatic activity of the type 2 methionine aminopeptidase (MetAP2), suggesting that MetAP2 is a specific target for both TNP-470 and ovalicin. To delineate the molecular interactions between the drugs and MetAP2, the enzyme bound forms of the drugs and the residue in MetAP2 that is covalently modified by the drugs will be identified and characterized. Generating a random MetAP2 mutant library and performing genetic screens will identify additional residue from MetAP2 that are involved in drug binding. To confirm that MetAP2 is the target for the drugs in endothelial cells both wild type and drug-resistant MetAP2 mutants will be expressed in endothelial cells to examine whether they confer upon the endothelial cells resistance to the drugs. Two of the most obvious effects of inhibition of MetAP2 is blockade of N-terminal myristoylation and alteration of rates of protein turnover among MetAP2 substrates, either of which can account for the inhibition of endothelial cell growth by the drugs. To identify relevant MetAP2 substrates, proteins will be detected and identified whose myristoylation or turnover are affected by the drugs by labeling whole cell proteins with (3H)-myristic acid and (35S)-methionine. Once such MetAP2 substrates are identified, their roles in mediating the action of the drugs will be further investigated in endothelial cells. The ultimate goal will be to achieve a complete understanding of mechanism of inhibition of endothelial cell growth by the drugs through identification of both direct and indirect mediators of the action of the drugs. Finally, fumagillin and ovalicin will be conjugated with cyclic RGD peptides to target the drugs to endothelial cells at tumor sites which are known to express high level of the RGD binding alpha-v-beta-3 integrin. These drug-RGD peptide conjugates may prove to be much more selective and less toxic than TNP-470 and related angiogenesis inhibitors.