Development of inhibitors for polyamine metabolism including cell death

为了阐明细胞死亡伴随细胞内多胺积累的机制，我们开展了以下几方面的研究工作：1)新的精胺/精胺-N^1-乙酰转移酶(SSAT)检测方法的建立和抑制SSAT活性的化合物的筛选；2)多胺类似物的合成及其对细胞生长和凋亡的影响；3)多胺对细胞体积和多胺分布的影响。结果表明，N1-去甲基亚硝胺作为荧光底物非常适用于重组人SSAT非放射性同位素SSAT检测方法。对重组SSAT蛋白及其在HTC细胞中的活性进行了动力学研究。结果表明，该方法适用于低水平生物样品的酶活性测定。从抑制剂的筛选中，我们发现双(烷基)去甲精胺是一种非常有效的化合物，其对SSAT的IC50远低于双(乙基)精胺，并且对多胺氧化具有抑制活性。本研究还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。细胞内多胺的积累改变了细胞体积，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.