鸟氨酸脱羧酶 (ornithine decarboxylase, ODC) 是多胺生物合成途径中的关键酶,其主要功能是催化鸟氨酸脱羧生成腐胺。在多种疾病和肿瘤细胞中,ODC的表达水平和催化活性都高于正常细胞,因此抑制ODC的活性是相关疾病预防和治疗的一个潜在途径。ODC抑制剂的发现和检验依赖于对其催化反应进程的监测,常采用的途径包括利用高效液相色谱法检测腐胺产量和利用同位素标记法检测二氧化碳的产量等。这些检测方法的繁琐操作和成本极大的限制了其应用,尤为突出的问题是这些方法很难实现高通量检测和实时检测。在本工作中,我们研究了基于大环分子葫芦脲 (cucurbituril, CB6) 与荧光染料DSMI (trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide) 的ODC酶活实时无标记检测法,系统分析了其应用范围和局限性,并对其进行了优化。最后,我们利用优化后的方法实现了对不同机制ODC抑制剂的活性评价。
Ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthetic pathway of polyamines, catalyzes the decarboxylation of ornithine. In many diseases and tumor cells, the expression level and the activity of ODC are generally higher than in normal cells. Therefore, inhibiting the activity of ODC is a potential approach for the prevention and treatment of many diseases including cancer. Monitoring the catalytic reaction of ODC is indispensable for discovering and evaluating ODC inhibitors. There are several commonly used methods for analyzing ODC’s activity, such as measuring the production of putrescine by high performance liquid chromatography, or quantifying the production of isotope labelled carbon dioxide. However, these methods are largely limited by their complicated manipulation and costs. More importantly, these methods are not impractical in real-time evaluation and high-throughput analyses. In this work, we systematically analyzed a real-time label-free evaluation method for ODC’s activity based on the macromolecule cucurbit  urea (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). We analyzed its limitations and optimized this method. Finally, the optimized method was effectively used in the evaluation of different types of ODC inhibitors with different inhibition mechanisms.