Discovery of amyloid-beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility.
Genetic and biochemical studies suggest that Alzheimer's disease (AD) is caused by a series of events initiated by the production and subsequent aggregation of the Alzheimer's amyloid beta peptide (Abeta), the so-called amyloid cascade hypothesis. Thus, a logical approach to treating AD is the development of small molecule inhibitors that either block the proteases that generate Abeta from its precursor (beta- and gamma-secretases) or interrupt and/or reverse Abeta aggregation. To identify potent inhibitors of Abeta aggregation, we have developed a high-throughput screen based on an earlier selection that effectively paired the folding quality control feature of the Escherichia coli Tat protein export system with aggregation of the 42-residue AD pathogenesis effecter Abeta42. Specifically, a tripartite fusion between the Tat-dependent export signal ssTorA, the Abeta42 peptide and the beta-lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the Abeta42 moiety. Here, we reasoned that small, cell-permeable molecules that inhibited Abeta42 aggregation would render the ssTorA-Abeta42-Bla chimera competent for Tat export to the periplasm where Bla is active against beta-lactam antibiotics such as ampicillin. Using a fluorescence-based version of our assay, we screened a library of triazine derivatives and isolated four nontoxic, cell-permeable compounds that promoted efficient Tat-dependent export of ssTorA-Abeta42-Bla. Each of these was subsequently shown to be a bona fide inhibitor of Abeta42 aggregation using a standard thioflavin T fibrillization assay, thereby highlighting the utility of our bacterial assay as a useful screen for antiaggregation factors under physiological conditions.
Lee
Li Ling
LL
Cornell University, Ithaca, New York 14853, USA.
Ha
HyungHo
H
Chang
Young-Tae
YT
DeLisa
Matthew P
MP
D000544:Alzheimer Disease / Q000235:genetics / Q000378:metabolism; D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism*; D002626:Chemistry, Pharmaceutical; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D011485:Protein Binding; D017510:Protein Folding; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries; D012995:Solubility; D014227:Triazines / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D016229:Amyloid beta-Peptides; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries; D014227:Triazines; C408585:twin-arginine translocase complex, E coli; D001618:beta-Lactamases