Garrabrant Thomas, Tuman Robert W, Ludovici Don, Tominovich Rose, Simoneaux Richard L, Galemmo Robert A, Johnson Dana L
Drug Discovery, Johnson & Johnson Pharmaceutical Research and Development, LLC, Spring House, Pennsylvania 19477, USA.
Angiogenesis. 2004;7(2):91-6. doi: 10.1007/s10456-004-6089-7.
The protein processing enzyme, methionine aminopeptidase-2 (MetAP-2), has been identified as a molecular target of fumagillin and its derivative, TNP-470, compounds known to inhibit endothelial cell proliferation and angiogenesis. A high-throughput screening program was undertaken to identify selective, reversible inhibitors of MetAP-2 in an attempt to discover structurally novel anti-angiogenic agents for potential therapeutic use in oncology. Approximately 90 small-molecule, reversible, selective inhibitors of rhMetAP-2 were identified. The most potent of these compounds contained a singly-substituted triazole moiety which exhibited an IC50 of 8 nM (95% confidence limits 5 to 13 nM) and was highly selective for MetAP-2 over MetAP-1 (approximately 60-fold difference in IC50 values). Unlike fumagillin, these MetAP-2 inhibitors failed to significantly inhibit growth factor-stimulated endothelial cell (HUVEC) proliferation or to suppress angiogenesis in the in vitro aortic ring explant model of microvessel outgrowth. The MetAP-2-inhibitory activity of these compounds was dependent on the divalent cation used as the metalloenzyme activating cofactor for MetAP-2. These inhibitors were identified using cobalt(II)-activated recombinant human MetAP-2 for screening compound libraries. When manganese (Mn2+) was substituted for cobalt following EDTA treatment and extensive dialysis of the MetAP-2 protein, these inhibitors were significantly less potent (40-fold increase in IC50) as inhibitors of MetAP-2. These results support the recent hypothesis that cobalt may not be the relevant divalent metal ion cofactor for MetAP-2 in cells and may explain the observed absence of cell-based activity using potent triazole inhibitors of cobalt-activated MetAP-2.
蛋白质加工酶甲硫氨酸氨肽酶-2（MetAP-2）已被确定为烟曲霉素及其衍生物TNP-470的分子靶点，这两种化合物已知可抑制内皮细胞增殖和血管生成。开展了一项高通量筛选计划，以鉴定MetAP-2的选择性、可逆抑制剂，试图发现结构新颖的抗血管生成剂，用于肿瘤学的潜在治疗。鉴定出了约90种小分子、可逆、选择性的重组人MetAP-2抑制剂。这些化合物中最有效的含有单取代三唑部分，其IC50为8 nM（95%置信限为5至13 nM），对MetAP-2的选择性远高于MetAP-1（IC50值相差约60倍）。与烟曲霉素不同，这些MetAP-2抑制剂未能显著抑制生长因子刺激的内皮细胞（HUVEC）增殖，也未能在体外微血管生长的主动脉环外植体模型中抑制血管生成。这些化合物的MetAP-2抑制活性取决于用作MetAP-2金属酶激活辅因子的二价阳离子。这些抑制剂是使用钴（II）激活的重组人MetAP-2筛选化合物文库鉴定出来的。在用EDTA处理并对MetAP-2蛋白进行广泛透析后，当用锰（Mn2+）替代钴时，这些抑制剂作为MetAP-2抑制剂的效力显著降低（IC50增加40倍）。这些结果支持了最近的假设，即钴可能不是细胞中MetAP-2的相关二价金属离子辅因子，这可能解释了使用钴激活的MetAP-2的有效三唑抑制剂时观察到的缺乏基于细胞的活性的现象。
