ABSTRACT
The Alzheimer's amyloid precursor protein (APP) is the metalloprotein that is cleaved to generate the pathogenic Abeta peptide. We showed that iron closely regulated the expression of APP by 5'-untranslated region (5'-UTR) sequences in APP mRNA. Iron modulated APP holoprotein expression by a pathway similar to iron control of the translation of the ferritin-L and -H mRNAs by iron-responsive elements in their 5'-UTRs. APP gene transcription is also responsive to copper deficit where the Menkes protein depleted fibroblasts of copper to suppress transcription of APP through metal regulatory and copper regulatory sequences upstream of the APP 5' cap site. APP is a copper-zinc metalloprotein and chelation of Fe(3+) by desferrioxamine and Cu(2+) by clioquinol appeared to provide effective therapy for the treatment of AD in limited patient studies. We have introduced an RNA-based screen for small APP 5'-UTR binding molecules to identify leads that limit APP translation (but not APLP-1 and APLP-2) and amyloid Abeta peptide production. A library of 1200 drugs was screened to identify lead drugs that limited APP 5'-UTR-directed translation of a reporter gene. The efficacy of these leads was confirmed for specificity in a cell-based secondary assay to measure the steady-state levels of APP holoprotein relative to APLP-1/APLP-2 by Western blotting. Several chelators were identified among the APP 5'-UTR directed leads consistent with the presence of an IRE stem-loop in front of the start codon of the APP transcript. The APP 5'-UTR-directed drugs--desferrioxamine (Fe(3+) chelator), tetrathiomolybdate (Cu(2+) chelator), and dimercaptopropanol (Pb(2+) and Hg(2+) chelator)--each suppressed APP holoprotein expression (and lowered Abeta peptide secretion). The novel anticholinesterase phenserine also provided "proof of concept" for our strategy to target the APP 5'-UTR sequence to identify "anti-amyloid" drugs. We further defined the interaction between iron chelation and phenserine action to control APP 5'-UTR-directed translation in neuroblastoma (SY5Y) transfectants. Phenserine was most efficient to block translation under conditions of intracellular iron chelation with desferrioxamine suggesting that this anticholinesterase operated through an iron (metal)-dependent pathway at the APP 5'-UTR site.
Subject(s)
5' Untranslated Regions/drug effects , Amyloid beta-Protein Precursor/genetics , Cholinesterase Inhibitors/pharmacology , Deferoxamine/pharmacology , Physostigmine/analogs & derivatives , Siderophores/pharmacology , Amyloid beta-Protein Precursor/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Humans , Neuroblastoma , Physostigmine/pharmacology , RNA, Messenger/chemistry , Transfection/methodsABSTRACT
We performed a screen for drugs that specifically interact with the 5' untranslated region of the mRNA coding for the Alzheimer's Amyloid Precursor Protein (APP). Using a transfection based assay, in which APP 5'UTR sequences drive the translation of a downstream luciferase reporter gene, we have been screening for new therapeutic compounds that already have FDA approval and are pharmacologically and clinically well-characterized. Several classes of FDA-pre-approved drugs (16 hits) reduced APP 5'UTR-directed luciferase expression (> 95% inhibition of translation). The classes of drugs include known blockers of receptor ligand interactions, bacterial antibiotics, drugs involved in lipid metabolism, and metal chelators. These APP 5'UTR directed drugs exemplify a new strategy to identify RNA-directed agents to lower APP translation and A beta peptide output for Alzheimer's disease therapeutics.
