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 (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.

Figure 1

Strategy for isolating Aβ42 aggregation inhibitors using the Tat pathway. (a) ssTorA-Aβ42-Bla expressed in the cytoplasm readily aggregates via Aβ42 self-assembly and thus is not exported by the Tat translocon that recognizes correctly folded proteins. (b) In the presence of small molecules that bind to Aβ42 and prevent aggregation, ssTorA-Aβ42-Bla remains soluble and is efficiently exported to the periplasm where Bla hydrolyzes Amp.

Figure 2

Amp resistance as an indicator of Aβ42 solubility. Inocula of (a) 1:100 or (b) 1:500 grown in the presence of varying Amp concentrations as indicated. Cells were induced to express ssTorA-Aβ42-Bla (white bars) or ssTorA-GM6-Bla (gray bars). All data was normalized to the growth rate of the 1:100-diluted cells expressing ssTorA-Aβ42-Bla in the absence of Amp. Data represents the average of six replicate experiments and the error bars represent the standard error of the mean (s.e.m.).

Figure 3

Fluorescence-based detection of Aβ42 aggregation in living E. coli cells using CCF2/AM. (a) Negative control cells expressing Sec-targeted cutinase (ssPhoA-CutA) from pBKPC and (b) positive control cells coexpressing ssPhoA-CutA and TEM-1 Bla with its native Sec signal peptide both from pBPC. Cells were incubated with 1 μM CCF2/AM for 1 h with gentle rocking and visualized by fluorescence microscopy (ex: 409 nm). (c) CCF2 fluorescence for cells expressing ssTorA-Aβ42-Bla or ssTorA-GM6-Bla in the presence (+) or absence (−) of cutinase as indicated. CCF2 fluorescence readings were normalized by dividing fluorescence values measured at 447 nm by those measured at 520 nm, and then normalized to OD₆₀₀ of the cells. All data was then normalized to the background fluorescence measured in cells expressing cutinase only. Data represents the average of three replicate experiments and the error bars represent the s.e.m.

Figure 4

Isolation of triazine derivatives that inhibit Aβ42 aggregation in E. coli. (a) Triazine scaffold where R₁ and R₂ substituents were varied to generate a combinatorial compound library. (b) R₁ and R₂ substituents for the four compounds identified as hits using the Aβ42 HTS CCF2/AM assay. (c) Compound 5B2 is structurally similar to positive hit 3B7 at the R₁ position but scored as an inactive compound in our cell-based screen.

Figure 5

In vitro characterization of aggregation inhibition using synthetic Aβ42. ThT fluorescence of synthetic Aβ42 fibrils after incubation of 20 μM Aβ42 with 10, 25, or 50 μM compounds as indicated. Tannic acid, a known inhibitor of Aβ42 aggregation, served as a positive control. 5B2 was structurally similar to our triazine hits but did not score as a hit in our screen and thus served as a negative control. 1E4, 3B7, 3G7, and 9D10 were compounds identified as Aβ42 aggregation inhibitors in the cell-based CCF2/AM screen of ∼1000 triazine derivatives. ThT fluorescence of Aβ42 incubated with triazine derivatives was expressed as a percentage of the DMSO-only negative control (note that the compounds were all dissolved in DMSO). Data represents the average of three replicate experiments and the error bars represent the s.e.m.