1887

Abstract

Spermidine/spermine-N1-acetyltransferase (SSAT) is a highly inducible and tightly controlled enzyme that is the rate-limiting step for polyamine catabolism. Because polyamines increase in dividing cells, SSAT has long been a target for cancer drug discovery. We have shown that the ability to enhance SSAT activity is a useful tool for cancer research, perhaps especially so for prostate cancer since the polyamine pathway is particularly active in this organ. SSAT can be induced by small molecules, including the biogenic polyamines themselves, which activate the negative feedback mechanism used to remove excess levels of these polyamines. SSAT activity is known to be affected by many mechanisms and good evidence indicates that translation control is important. We recently found that SSAT translation control involves a stem loop at the beginning of the open reading frame (ORF) and an upstream ORF 5' to the initiating methionine. A nucleolin isoform suppresses translation by binding to and stabilizing a 5' stem loop of SSAT mRNA but this repression is released when increased polyamines activate nucleolin autocatalysis; i.e., this is a negative feedback system. The current study is designed to exploit these exciting results with the overall goal to identify drug candidates with high potency and desirable physical properties to take forward into pre-clinical development. Here we report the development and validation of a luciferase-based reporter system for the identification of compounds that are able to promote the translation of SSAT. The translational de-repression sensor system, which uses HEK293T cells transformed with a construct composed of SSAT mRNA modified to lack uORF function, is mutated to overcome repression and is linked at the 3' end with luciferase. As a proof of principle of the utility of the SSAT translation sensor, we screened 2 libraries, the Redox library consisting of antioxidants (84 compounds) and the FDA Approved Drug library (1,200 compounds). Untreated cells were used as the negative control, and cells treated with 10 µM DENSPM (N1, N11-diethylnorspermine) were the positive control. Twelve compounds were identified from these libraries that activate SSAT translation by at least 35% more than the basal expression. We conclude that an SSAT translational control-based high throughput screening sensor can lead to the identification of novel pharmacophores that are able to prevention and/or treat prostate cancer.

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/content/papers/10.5339/qfarf.2012.AESNP8
2012-10-01
2020-07-05
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