Discovery of amyloid-beta aggregation inhibitors using an engineered assay for intracellular protein folding and solubility

Protein Sci. 2009 Feb;18(2):277-86. doi: 10.1002/pro.33.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alzheimer Disease / genetics
  • Alzheimer Disease / metabolism
  • Amyloid beta-Peptides / genetics
  • Amyloid beta-Peptides / metabolism*
  • Chemistry, Pharmaceutical
  • Drug Evaluation, Preclinical / methods*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism
  • Fluorescent Dyes
  • Membrane Transport Proteins / genetics
  • Membrane Transport Proteins / metabolism
  • Microscopy, Fluorescence
  • Models, Molecular
  • Protein Binding
  • Protein Folding*
  • Protein Sorting Signals / genetics
  • Protein Sorting Signals / physiology
  • Protein Structure, Quaternary
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Reproducibility of Results
  • Small Molecule Libraries
  • Solubility*
  • Triazines / metabolism
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism

Substances

  • Amyloid beta-Peptides
  • Escherichia coli Proteins
  • Fluorescent Dyes
  • Membrane Transport Proteins
  • Protein Sorting Signals
  • Recombinant Fusion Proteins
  • Small Molecule Libraries
  • Triazines
  • twin-arginine translocase complex, E coli
  • beta-Lactamases