INHIBITION OF ANGIOGENESIS BY TNP 470 AND OVALICIN

描述：这项提议的主要目标是获得一种分子 对天然药物抑制血管生成机制的认识 产品烟青素、卵黄素和合成类似物TNP-470，以及 通过靶向提高这些药物的疗效和减少副作用 转移到肿瘤上。最近研究表明，TNP-470和卵黄素 有效抑制2型蛋氨酸的酶活性 氨基肽酶(MetAP2)，提示MetAP2是 TNP-470和Ovalicin。来描述分子间的相互作用 药物和MetAP2，药物的酶结合形式和残留物 被药物共价修饰的MetAP2将被识别并 特色化的。生成随机MetAP2突变体文库并执行 基因筛查将识别来自MetAP2的额外残基 牵涉到药物结合。确认MetAP2是 内皮细胞中的药物野生型和耐药MetAP2突变体 将在内皮细胞中表达，以检测它们是否赋予 血管内皮细胞对药物产生抗药性。其中最明显的两个 抑制MetAP2的作用是阻断N-末端肉豆蔻化和 MetAP2底物之间蛋白质周转率的变化 这可能是血管内皮细胞生长受到抑制的原因 毒品。为了确定相关的MetAP2底物，将检测蛋白质 并确定哪些人的肉豆蔻化或周转受到药物的影响 用(~3H)-肉豆蔻酸和(35S)-蛋氨酸标记全细胞蛋白。 一旦确定了这样的MetAP2底物，它们在调节 这些药物的作用将在内皮细胞中进一步研究。这个 最终的目标将是达到一个完整的机制的理解 通过鉴定药物抑制内皮细胞的生长 药物作用的直接和间接介体。最后， 将烟青素和卵黄素与环状RGD多肽偶联， 这些药物针对肿瘤部位的内皮细胞，这些细胞已知 表达高水平的RGD结合α-v-β-3整合素。这些 药物-RGD多肽结合物可能被证明具有更高的选择性和更低的选择性 毒性超过TNP-470和相关的血管生成抑制剂。

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.