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

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 (A beta), 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 A beta from its precursor (beta- and gamma-secretases) or interrupt and/or reverse A beta aggregation. To identify potent inhibitors of A beta 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 A beta 42. Specifically, a tripartite fusion between the Tat-dependent export signal ssTorA, the A beta 42 peptide and the beta-lactamase (Bla) reporter enzyme was found to be export incompetent due to aggregation of the A beta 42 moiety. Here, we reasoned that small, cell-permeable molecules that inhibited A beta 42 aggregation would render the ssTorA-A beta 42-Bla chimera competent for Tat export to the periplasm where Bla is active against b- 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-A beta 42-Bla. Each of these was subsequently shown to be a bona fide inhibitor of A beta 42 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.