Department of Biochemistry and Fels Institute for Cancer Research, School of Medicine and 2 Moulder Center for Drug Discovery, School of Pharmacy, Temple University 3307 N. Broad Street, Philadelphia, PA 19140.
Curr Pharm Des. 2014;20(2):245-52. doi: 10.2174/13816128113199990035.
Spermidine/spermine-N1-acetyltransferase (SSAT) is a mitochondrial-localized enzyme that is highly inducible and tightly controlled and is the rate-limiting enzyme in polyamine catabolism. It is known that SSAT is induced when polyamine level increases. Although multiple mechanisms have been implicated, translational control is thought to be paramount. Previous studies with transgenic and knockout mice suggested that for certain human conditions, the modulation of SSAT levels could offer therapeutic benefits. Besides polyamines and their analogs, certain stimuli can increase SSAT levels, suggesting that the development of reporters for high throughput screening can lead to the identification of novel pharmacophores that can modulate SSAT translation. Here we report the development and validation of a luciferase-based biosensor system for the identification of compounds that are able to either promote or prevent the translation of SSAT. The system uses HEK293T cells transfected with a construct composed of SSAT mRNA modified to lack upstream open reading frame (uORF) function, is mutated to reduce translational repression and is linked with luciferase. As a proof of principle of the utility of the SSAT translation sensor, we screened the Prestwick drug library (1,200 FDA Approved compounds). The library contained 15 compounds that activated SSAT translation by at least 40% more than the basal expression, but none exceeded the positive control N1, N11-diethylnorspermine. On the other hand, 38 compounds were found to strongly inhibit SSAT translation. We conclude that this biosensor can lead to the identification of novel pharmacophores that are able to modulate the translation of SSAT.
精脒/精胺-N1-乙酰基转移酶（SSAT）是一种定位于线粒体的酶，具有高度诱导性和严格的调控性，是多胺分解代谢的限速酶。已知当多胺水平增加时，SSAT 会被诱导。尽管已经涉及多种机制，但翻译控制被认为是最重要的。以前使用转基因和敲除小鼠的研究表明，对于某些人类疾病，调节 SSAT 水平可能会带来治疗益处。除了多胺及其类似物外，某些刺激物可以增加 SSAT 水平，这表明开发高通量筛选的报告基因可以导致鉴定出能够调节 SSAT 翻译的新型药效团。在这里，我们报告了一种基于荧光素酶的生物传感器系统的开发和验证，用于鉴定能够促进或阻止 SSAT 翻译的化合物。该系统使用转染了由 SSAT mRNA 构建的 HEK293T 细胞，该构建体被修饰为缺乏上游开放阅读框（uORF）功能，突变以减少翻译抑制，并与荧光素酶相连。作为 SSAT 翻译传感器实用性的原理证明，我们筛选了 Prestwick 药物库（1200 种 FDA 批准的化合物）。该库包含 15 种化合物，它们使 SSAT 翻译的表达比基础表达增加至少 40%，但没有一种化合物超过阳性对照 N1、N11-二乙基-norspermine。另一方面，发现 38 种化合物强烈抑制 SSAT 翻译。我们得出结论，这种生物传感器可以导致鉴定出能够调节 SSAT 翻译的新型药效团。
