Development of inhibitors for polyamine metabolism including cell death

为了阐明多胺在细胞中积累伴随的细胞死亡机制，已经进行了以下研究以开发亚精胺/精胺-N ^1-乙酰转移酶（SSAT）的强抑制剂：1）开发新的SSAT测定方法和筛选抑制SSAT活性的化合物的测定方法，2）多胺类似物的合成和它们对培养的细胞系中的细胞生长或凋亡的作用的检查，（3）多胺积累后细胞体积和多胺分布的变化。使用重组人SSAT的放射性同位素SSAT测定方法。在HTC细胞中对重组SSAT蛋白和细胞SSAT活性进行动力学研究。结果表明，该方法适用于低酶含量的生物样品中酶活性的测定。通过对抑制剂的筛选，我们发现双（烷基）去甲精脒是一种很强的抑制剂，其对SSAT活性的IC_（50）远低于双（乙基）精胺，对多胺氧化酶具有抑制活性，并发现所合成的化合物具有抑制细胞生长或诱导细胞凋亡的作用，用Coulter计数器检测细胞体积和多胺分布的变化，超滤法，分别与亚精胺积累的细胞。这表明，细胞内多胺的积累使细胞体积缩小，改变了多胺的分布。

Aiming at elucidation of the mechanism of cell death accompanied by the polyamine accumulation in cells, the following research has been done to develop strong inhibitor for spermidine/spermine-N^1-acetyltransferase (SSAT): 1) Development of novel assay method for SSAT and screening assay method for compounds inhibiting SSAT activity, 2) Synthesis of polyamine analogues and examination of their effect on cell growth or apoptosis in cultured cell lines, 3) Changes in cell volume and polyamine distribution in cells accumulated with polyamine.It was found that N1-dansylnorspermine was very useful as a fluorescent substrate for non-radio isotopic SSAT assay method using recombinant human SSAT. The kinetic studies were performed for the recombinant SSAT protein and cellular SSAT activity in HTC cells. It was shown that the method was useful for measuring the activity in biological sample having low level of the enzyme. From the screening for the inhibitor, we found that bis(alkyl)norspermidine was a very potent one, whose IC50 to SSAT activity was much lower than bis(ethyl)spermine, and had inhibition activity for polyamine oxidase.It was also found that the compounds synthesized in this study were effective for cell growth inhibition or induction of apoptosis.The changes in cell volume and distribution of polyamine were examined by Coulter counter and ultrafiltration method, respectively, for the cells accumulated with spermidine. It was suggested that the cell volume was decreased and the distribution of polyamine was changed by the accumulation of cellular polyamine.