HTS Molecules Targeting APP 5'untranslated Region(RMI)

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The 5'untranslated region of the mRNAforthe Alzheimer's Amyloid Precursor Protein (APP 5'UTR) is a key translational regulatory element that sets the amount of APP production in any given cell. IL-1 enhanced the interaction between Iron-regulatory Proteins (IRPs) and APP 5'UTR RNA secondary structure via an Iron- responsive Element (IRE). This regulation suggested the APP 5'UTR to be an excellent drug target. Our laboratory gained valuable experience in the field of drug discovery when we screened a library of FDA- pre-approved drugs and identified 17 leads that limited APP 5'UTR driven translation using transiently transfected neuroblastoma cells. In secondary western blot-based assays paroxetine (SSRI) and dimercaptopropanol (chelator) selectivley reduced APP holoprotein expression. As proof of drug selectivity achieved during the APP 5'UTR screen these two hits did not change APLP-1 and APLP-2 expression in SH-SY5Y cells (Payton et al.,2003). A pilot study indicated that Paroxetine reduced the amyloid burden in a transgenic mouse model for AD (TgCRNDS mice). The data to be gathered through this R03 RFA mechanism will assist us to develop our FDA pilot screen to set up a high throughput transfection based screen of an important RNA target. Use of automated equipment for 384 well based screens is now available through our collaboration with the Laboratory of Drug Discovery for Neurodegenreration (LDDN). Our strategy will be to optimize a high throughput screen of the 110,000 compounds in the LDDN drug library to identify new agents that selectively inhibit translation driven by the APP 5'UTR enhancer. These leads may well provide downstream therapeutic anti-amyloid limiting action. Use of our drug hits in RNA gel-shift and APP expression based experiments will probe the pathway of APP translation. IRP-1 and IRP-2 post-transcriptionally control iron homeostasis by modulating ferritin translation and transferrin receptor mRNA stability. HTS hits directed to the 146 nt APP 5'UTR will provide new tools to assess precisely the importance of the interaction of IRP-1 and IRP-2 with 5'UTR of the mRNA encodiing APP (metalloprotein), relative to the mRNAs of other key iron proteins.